238P1B2 protein

ABSTRACT

A novel gene (designated 238P1B2) and its encoded protein, and variants thereof, are described wherein 238P1B2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 238P1B2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 238P1B2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 238P1B2 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 10/114,669, filed Apr. 1, 2002, the contents of which is hereby incorporated by reference herein in its entirety.

SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs are incorporated herein by reference in its entirety: A compact disc copy of the Sequence Listing (COPY 1) (file name: 5115820065.txt, date recorded: Sep. 3, 2002, size: 1,448 KB); a duplicate compact disc copy of Sequence Listing (COPY 2) (file name: 5115820065.txt, date recorded: Sep. 3, 2002, size: 1,448 KB); a computer readable form copy of the Sequence Listing (CRF COPY) (file name: 5115820065.txt, date recorded: Sep. 3, 2002, size: 1,448 KB).

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encoded protein, termed 238P1B2, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 238P1B2.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 238P1B2, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 238P1B2 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 238P1B2 are provided. The tissue-related profile of 238P1B2 in normal adult tissues, combined with the over-expression observed in the tumors listed in Table I, shows that 238P1B2 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary to all or part of the 238P1B2 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 238P1B2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 238P1B2-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 238P1B2 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 238P1B2 genes, mRNAs, or to 238P1B2-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 238P1B2. Recombinant DNA molecules containing 238P1B2 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 238P1B2 gene products are also provided. The invention further provides antibodies that bind to 238P1B2 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 238P1B2 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 238P1B2. A typical embodiment of this invention provides methods for monitoring 238P1B2 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 238P1B2 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 238P1B2 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 238P1B2 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 238P1B2. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 238P1B2 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 238P1B2 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 238P1B2 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 238P1B2. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 238P1B2 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 238P1B2 production) or a ribozyme effective to lyse 238P1B2 mRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 238P1B2 SSH sequence of 210 nucleotides (SEQ ID: 8869).

FIG. 2. The cDNA (SEQ ID: 8870) and amino acid sequence (SEQ ID: 8871) of 238P1B2 variant 1A is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acids 2133 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8872) and amino acid sequence (SEQ ID: 8873) of 238P1B2 variant 1B is shown in FIG. 2B. The open reading frame extends from nucleic acid 1947 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8874) and amino acid (SEQ ID: 8875) sequence of 238P1B2 variant 2 is shown in FIG. 2C; the codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2133 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8876) and amino acid (SEQ ID: 8877) sequence of 238P1B2 variant 3 is shown in FIG. 2D, the codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2133 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8878) and amino acid (SEQ ID: 8879) sequence of 238P1B2 variant 4 is shown in FIG. 2E; the codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2133 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8880) and amino acid (SEQ ID: 8881) sequence of 238P1B2 variant 5 is shown in FIG. 2F; the codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2133 to 2897 including the stop codon. The nucleic acid (SEQ ID: 8882) and amino acid (SEQ ID: 8883) sequence of 238P1B2 variant 6 is shown in FIG. 2G, the codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2133 to 2897 including the stop codon.

FIG. 3. Amino acid sequence of 238P1B2 variant 1A (SEQ ID: 8884) is shown in FIG. 3A; it has 254 amino acids. The amino acid sequence of 238P1B2 variant 1B (SEQ ID: 8885) is shown in FIG. 3B; it has 316 amino acids. The amino acid sequence of 238P1B2 variant 2 (SEQ ID: 8886) is shown in FIG. 3C; it has 254 amino acids.

FIG. 4. A. Nucleic Acid sequence alignment of 238P1B2 variant 1 (SEQ ID: 8887) with mouse olfactory receptor MOR14-1 (SEQ ID: 8888). B. Nucleic Acid sequence alignment of 238P1B2 variant 1 (SEQ ID: 8889) with mouse olfactory receptor MOR14-10 (SEQ ID: 8890). C. Amino Acid sequence alignment of 238P1B2 v.1A (SEQ ID: 8891) with mouse olfactory receptor MOR14-1 (SEQ ID: 8892). D. Amino Acid sequence alignment of 238P1B2 v.1A (SEQ ID: 8893) with prostate specific GPCR PHOR-1 (SEQ ID: 8894). E. Amino acid sequence alignment of 238P1B2 variant I (SEQ ID: 8895) with human olfactory receptor 5112 (SEQ ID: 8896). F. Amino Acid sequence Clustal Alignment of the three 238P1B2 variants (238P1B2 v.1A (SEQ ID: 8897); 238P1B2 v.1B (SEQ ID: 8898); 238P1B2 v.2 (SEQ ID: 8899)), depicting that 238P1B2 V1B contains an additional 62 aa at its N-terminus relative to V1A, and that 238P1B2 V2 carries a I to T point mutation at aa 225 relative to V1A

FIG. 5. Hydrophilicity amino acid profile of A) 238P1B2 and B) 238P1B2 var1A determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website on the world wide web through the ExPasy molecular biology server.

FIG. 6. Hydropathicity amino acid profile of A) 238P1B2 and B) 238P1B2 var1A determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website on the world wide web through the ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of A) 238P1B2 and B) 238P1B2 var1A determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website on the world wide web through the ExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of A) 238P1B2 and B) 238P1B2 var1A determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website on the world wide web (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of A) 238P1B2 and B) 238P1B2 var1A determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website through the ExPasy molecular biology server.

FIG. 10. Schematic display of nucleotide variants of 238P1B2. Variant 238P1B2 v.2 through 238P1B2 v.6 are variants with single nucleotide variations. Black box shows the same sequence as 238P1B2 v.1. Numbers correspond to those of 238P1B2 v.1. SNPs are indicated above the box.

FIG. 11. Schematic display of protein variants of 238P1B2. Nucleotide variant 238P1B2 v.1 in FIG. 10 codes for protein variants 238P1B2 v.1A and v.1B. Nucleotide variant 238P1B2 v.2 in FIG. 10 codes for protein variant 238P1B2 v.2. Black box shows the same sequence as 238P1B2 v.1. Numbers in “( )” underneath the box correspond to those of 238P1B2 v.1A. Single amino acid differences are indicated above the box.

FIG. 12. A, B. Secondary structure prediction for 238P1B2 variant 1a (SEQ ID: 8901) and variant1b (SEQ ID: 8902). The secondary structures of 238P1B2 variant 1a (A) and of variant 1b (B) proteins were predicted using the HNN—Hierarchical Neural Network method (Guermeur, 1997), accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed for each variant.

C, D, E, F. Transmembrane prediction for 238P1B2 variant 1a and 1b. C, E. Schematic representations of the probability of existence of transmembrane regions and orientation of 238P1B2 variant 1a (C) and variant 1b (E) based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). D, F. Schematic representation of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 238P1B2 variant 1a (D) and variant 1b (F) based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server. The results of the transmembrane prediction programs presented depict 238P1B2 variant 1a as most likely containing 6 transmembrane domains and variant 1b 7 transmembrane domains.

FIG. 13: Probable topology and structure of 238P1B2 variants 1 and 1B.

FIG. 14. Expression of 238P1B2 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), and prostate cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 238P1B2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 238P1B2 in prostate cancer pool but not in vital pool 1 and vital pool 2.

FIG. 15. Expression of 238P1B2 in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 μg of mRNA/lane were probed with the 238P1B2 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show absence of 238P1B2 expression in all 16 normal tissues tested.

FIG. 16. Expression of 238P1B2 in Multiple Normal Tissues. An mRNA dot blot containing 76 different samples from human tissues was analyzed using a 238P1B2 SSH probe. Expression was only detected in placenta.

FIG. 17. Expression of 238P1B2 in Human Patient Cancer Specimens and Normal Tissues. RNA was extracted from a pool of three prostate cancer patient specimens, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC), normal lung (NL), normal breast (NBr), and normal ovary (NO. Northern blot with 10 μg of total RNA/lane was probed with 238P1B2 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 238P1B2 in the prostate cancer pool and normal ovary.

FIG. 18. Expression of 238P1B2 in prostate cancer patient tissues. RNA was extracted from normal prostate (N), prostate cancer xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer cell lines (LNCaP and PC3), and prostate cancer patient tumors (T). Northern blots with 10 μg of total RNA were probed with the 238P1B2 SSH fragment. Size standards in kilobases are on the side. Results show strong expression of 238P1B2 in prostate tumor tissues. The lower panel represents the ethidium bromide staining of the gel.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 238P1B2 Polynucleotides

-   -   II.A.) Uses of 238P1B2 Polynucleotides         -   II.A.1.) Monitoring of Genetic Abnormalities         -   II.A.2.) Antisense Embodiments         -   II.A.3.) Primers and Primer Pairs         -   II.A.4.) Isolation of 238P1B2-Encoding Nucleic Acid             Molecules         -   II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector             Systems

III.) 238P1B2-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments     -   III.B.) Expression of 238P1B2-related Proteins     -   III.C.) Modifications of 238P1B2-related Proteins     -   III.D.) Uses of 238P1B2-related Proteins

IV.) 238P1B2 Antibodies

V.) 238P1B2 Cellular Immune Responses

VI.) 238P1B2 Transgenic Animals

VII.) Methods for the Detection of 238P1B2

VIII.) Methods for Monitoring the Status of 238P1B2-related Genes and Their Products

IX.) Identification of Molecules That Interact With 238P1B2

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines     -   X.B.) 238P1B2 as a Target for Antibody-Based Therapy     -   X.C.) 238P1B2 as a Target for Cellular Immune Responses         -   X.C.1. Minigene Vaccines         -   X.C.2. Combinations of CTL Peptides with Helper Peptides         -   X.C.3. Combinations of CTL Peptides with T Cell Priming             Agents         -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL             and/or HTL Peptides     -   X.D.) Adoptive Immunotherapy     -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic         Purposes

XI.) Diagnostic and Prognostic Embodiments of 238P1B2.

XII.) Inhibition of 238P1B2 Protein Function

-   -   XII.A.) Inhibition of 238P1B2 With Intracellular Antibodies     -   XII.B.) Inhibition of 238P1B2 with Recombinant Proteins     -   XII.C.) Inhibition of 238P1B2 Transcription or Translation     -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) KITS

I.) Definitions:

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 238P1B2 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 238P1B2. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 238P1B2-related protein). For example an analog of a 238P1B2 protein can be specifically bound by an antibody or T cell that specifically binds to 238P1B2.

The term “antibody” is used in the broadest sense. Therefore an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-238P1B2 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-238P1B2 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-238P1B2 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”

The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 238P1B2 genes or that encode polypeptides other than 238P1B2 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 238P1B2 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 238P1B2 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 238P1B2 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 238P1B2-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.

The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymine (T), as shown for example in FIG. 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind or interact with 238P1B2, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 238P1B2 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 238P1B2 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 238P1B2 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.

The “238P1B2-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 238P1B2 proteins or fragments thereof, as well as fusion proteins of a 238P1B2 protein and a heterologous polypeptide are also included. Such 238P1B2 proteins are collectively referred to as the 238P1B2-related proteins, the proteins of the invention, or 238P1B2. The term “238P1B2-related protein” refers to a polypeptide fragment or an 238P1B2 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.

II.) 238P1B2 Polynucleotides

One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 238P1B2 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 238P1B2-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 238P1B2 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 238P1B2 gene, mRNA, or to an 238P1B2 encoding polynucleotide (collectively, “238P1B2 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.

Embodiments of a 238P1B2 polynucleotide include: a 238P1B2 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 238P1B2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 238P1B2 nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or         consisting of a sequence as shown in FIG. 2, wherein T can also         be U;     -   (II) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2A, from nucleotide         residue number 2133 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (III) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2B, from nucleotide         residue number 1947 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (IV) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2C, from nucleotide         residue number 2133 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (V) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2D, from nucleotide         residue number 2133 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (VI) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2E, from nucleotide         residue number 2133 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (VII) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2F, from nucleotide         residue number 2133 through nucleotide residue number 2894,         followed by a stop codon, wherein T can also be U;     -   (VIII) a polynucleotide comprising, consisting essentially of,         or consisting of the sequence as shown in FIG. 2G, from         nucleotide residue number 2133 through nucleotide residue number         2894, followed by a stop codon, wherein T can also be U;     -   (IX) a polynucleotide that encodes an 238P1B2-related protein         that is at least 90% homologous to an entire amino acid sequence         shown in FIG. 2A-G;     -   (X) a polynucleotide that encodes an 238P1B2-related protein         that is at least 90% identical to an entire amino acid sequence         shown in FIG. 2A-G;     -   (XI) a polynucleotide that encodes at least one peptide set         forth in Tables V-XVIII or Table XIX;     -   (XII) a polynucleotide that encodes a peptide region of at least         5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 254 that includes an amino acid position having         a value greater than 0.5 in the Hydrophilicity profile of FIG.         5A, or of FIG. 3B in any whole number increment up to 316 that         includes an amino acid position having a value greater than 0.5         in the Hydrophilicity profile of FIG. 5B;     -   (XIII) a polynucleotide that encodes a peptide region of at         least S amino acids of a peptide of FIG. 3A in any whole number         increment up to 254 that includes an amino acid position having         a value less than 0.5 in the Hydropathicity profile of FIG. 6A,         or of FIG. 3B in any whole number increment up to 316, that         includes an amino acid position having a value less than 0.5 in         the Hydropathicity profile of FIG. 6B;     -   (XIV) a polynucleotide that encodes a peptide region of at least         S amino acids of a peptide of FIG. 3A in any whole number         increment up to 254 that includes an amino acid position having         a value greater than 0.5 in the Percent Accessible Residues         profile of FIG. 7A, or of FIG. 3B in any whole number increment         up to 316, that includes an amino acid position having a value         greater than 0.5 in the Percent Accessible Residues profile of         FIG. 7B;     -   (XV) a polynucleotide that encodes a peptide region of at least         5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 254 that includes an amino acid position having         a value greater than 0.5 in the Average Flexibility profile on         FIG. 8A, or of FIG. 3B in any whole number increment up to 316,         that includes an amino acid position having a value greater than         0.5 in the Average Flexibility profile on FIG. 8B;     -   (XVI) a polynucleotide that encodes a peptide region of at least         5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 254 that includes an amino acid position having         a value greater than 0.5 in the Beta-turn profile of FIG. 9A, or         of FIG. 3B in any whole number increment up to 316, that         includes an amino acid position having a value greater than 0.5         in the Beta-turn profile of FIG. 9B;     -   (XVII) a polynucleotide that encodes a 238P1B2-related protein         whose sequence is encoded by the cDNAs contained in the plasmid         deposited with American Type Culture Collection as Accession No.         PTA-4124 on Mar. 7, 2002;     -   (XVIII) a polynucleotide that is fully complementary to a         polynucleotide of any one of (I)—(XVII);     -   (XIX) a polynucleotide that selectively hybridizes under         stringent conditions to a polynucleotide of (I)-(XVIII);     -   (XX) a peptide that is encoded by any of (I)-(XIX); and, (XXI) a         polynucleotide of any of (I)-(XIX) or peptide of (XX) together         with a pharmaceutical excipient and/or in a human unit dose         form.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 238P1B2 polynucleotides that encode specific portions of 238P1B2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, or 254 contiguous amino acids of 238P1B2;

(b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, or 254 contiguous amino acids of variant 1A;

(c) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, or 316 contiguous amino acids of variant 1B; or

(d) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, or 254 contiguous amino acids of variant 2.

For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3, or encoding regions from about amino acid 100 to amino acids later in the sequence, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100 through the carboxyl terminal amino acid of the 238P1B2 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 238P1B2 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid I (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 238P1B2 protein or variants shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 238P1B2 sequence or variants as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed herein include 238P1B2 polynucleotide fragments encoding one or more of the biological motifs contained within a 238P1B2 protein sequence or variant sequence, including one or more of the motif-bearing subsequences of a 238P1B2 protein or variant set forth in Tables V-XVIII and Table XIX.

Note that to determine the starting position of any peptide set forth in Tables V-XVIII and Table XIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed at the beginning of Table XIX. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed at the beginning of Table XIX. Accordingly if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables V-XVIII and Table XIX to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables V-XVIII and Table XIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables V-XVIII and at least once in Table XIX, or an oligonucleotide that encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes which comprise a peptide region, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9.

In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 238P1B2 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 238P1B2 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of 238P1B2 Polynucleotides

II.A.I.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 238P1B2 gene maps to the chromosomal location set forth in Example 3. For example, because the 238P1B2 gene maps to this chromosome, polynucleotides that encode different regions of the 238P1B2 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 238P1B2 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 238P1B2 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 238P1B2 was shown to be highly expressed in bladder and other cancers, 238P1B2 polynucleotides are used in methods assessing the status of 238P1B2 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 238P1B2 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 238P1B2 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 238P1B2. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 238P1B2 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 238P1B2. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 238P1B2 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 238P1B2 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The 238P1B2 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 238P1B2 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 238P1B2 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 238P1B2 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 238P1B2 mRNA. Optionally, 238P1B2 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 238P1B2. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 238P1B2 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 238P1B2 polynucleotide in a sample and as a means for detecting a cell expressing a 238P1B2 protein.

Examples of such probes include polypeptides comprising all or part of the human 238P1B2 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 238P1B2 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 238P1B2 mRNA.

The 238P1B2 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 238P1B2 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 238P1B2 polypeptides; as tools for modulating or inhibiting the expression of the 238P1B2 gene(s) and/or translation of the 238P1B2 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 238P1B2 or 238P1B2 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 238P1B2-Encoding Nucleic Acid Molecules

The 238P1B2 cDNA sequences described herein enable the isolation of other polynucleotides encoding 238P1B2 gene product(s), as well as the isolation of polynucleotides encoding 238P1B2 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 238P1B2 gene product as well as polynucleotides that encode analogs of 238P1B2-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 238P1B2 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 238P1B2 gene cDNAs can be identified by probing with a labeled 238P1B2 cDNA or a fragment thereof. For example, in one embodiment, a 238P1B2 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 238P1B2 gene. A 238P1B2 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 238P1B2 DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containing an 238P1B2 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 238P1B2 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 238P1B2 or a fragment, analog or homolog thereof can be used to generate 238P1B2 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 238P1B2 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11: 1785). Using these expression vectors, 238P1B2 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 238P1B2 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 238P1B2 and 238P1B2 mutations or analogs.

Recombinant human 238P1B2 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 238P1B2-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 238P1B2 or fragment, analog or homolog thereof, a 238P1B2-related protein is expressed in the 293T cells, and the recombinant 238P1B2 protein is isolated using standard purification methods (e.g., affinity purification using anti-238P1B2 antibodies). In another embodiment, a 238P1B2 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 238P1B2 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 238P1B2 coding sequence can be used for the generation of a secreted form of recombinant 238P1B2 protein.

As discussed herein, redundancy in the genetic code permits variation in 238P1B2 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 238P1B2-related Proteins

Another aspect of the present invention provides 238P1B2-related proteins. Specific embodiments of 238P1B2 proteins comprise a polypeptide having all or part of the amino acid sequence of human 238P1B2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 238P1B2 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 238P1B2 shown in FIG. 2 or FIG. 3.

In general, naturally occurring allelic variants of human 238P1B2 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 238P1B2 protein contain conservative amino acid substitutions within the 238P1B2 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 238P1B2. One class of 238P1B2 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 238P1B2 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 238P1B2 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 238P1B2 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 238P1B2 variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 238P1B2 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 238P1B2 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to an 238P1B2 variant also specifically binds to a 238P1B2 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 238P1B2 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 238P1B2-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 238P1B2 protein variants or analogs comprise one or more of the 238P1B2 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 238P1B2 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 238P1B2 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 238P1B2 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid I to about amino acid 10 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 238P1B2 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 238P1B2 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 238P1B2 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

238P1B2-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 238P1B2-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 238P1B2 protein (or variants, homologs or analogs thereof).

III.A.) Motif-bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed herein include 238P1B2 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 238P1B2 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., Epimatrix™ and Epimer™, Brown University, and BIMAS).

Motif bearing subsequences of all 238P1B2 variant proteins are set forth and identified in Tables V-XVIII and Table XIX.

Table XX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.

Polypeptides comprising one or more of the 238P1B2 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 238P1B2 motifs discussed above are associated with growth dysregulation and because 238P1B2 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XVIII and Table XIX. CTL epitopes can be determined using specific algorithms to identify peptides within an 238P1B2 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, and BIMAS). Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 9733602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al, 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table XXI, and/or, one or more of the polypeptides comprising combinations of the different motifs set forth in Table XIX, and/or, one or more of the predicted CTL epitopes of Table V through Table XVIII, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

238P1B2-related proteins are embodied in many forms, preferably in isolated form. A purified 238P1B2 protein molecule will be substantially free of other proteins or molecules that impair the binding of 238P1B2 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 238P1B2-related proteins include purified 238P1B2-related proteins and functional, soluble 238P1B2-related proteins. In one embodiment, a functional, soluble 238P1B2 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 238P1B2 proteins comprising biologically active fragments of a 238P1B2 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 238P1B2 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 238P1B2 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.

238P1B2-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-238P1B2 antibodies, or T cells or in identifying cellular factors that bind to 238P1B2. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within an 238P1B2 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, and BIMAS). Illustrating this, peptide epitopes from 238P1B2 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII, Table XIX). Specifically, the complete amino acid sequence of the 238P1B2 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS); in addition to the site SYFPEITHI.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 238P1B2 predicted binding peptides are shown in Tables V-XVIII and Table XIX herein. In Tables V-XVIII and Table XIX, selected candidates, 9-mers, 10-mers, and 15-mers for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV are to be “applied” to a 238P1B2 protein in accordance with the invention. As used in this context “applied” means that a 238P1B2 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 238P1B2 protein of 8, 9, 10, or II amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 238P1B2-related Proteins

In an embodiment described in the examples that follow, 238P1B2 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 238P1B2 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 238P1B2 protein in transfected cells. The secreted HIS-tagged 238P1B2 in the culture media can be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 238P1B2-related Proteins

Modifications of 238P1B2-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 238P1B2 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 238P1B2 protein. Another type of covalent modification of a 238P1B2 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 238P1B2 comprises linking a 238P1B2 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 238P1B2-related proteins of the present invention can also be modified to form a chimeric molecule comprising 238P1B2 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 238P1B2 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 238P1B2. A chimeric molecule can comprise a fusion of a 238P1B2-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a238P1B2 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 238P1B2-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 238P1B2 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 238P1B2-related Proteins

The proteins of the invention have a number of different specific uses. As 238P1B2 is highly expressed in prostate and other cancers, 238P1B2-related proteins are used in methods that assess the status of 238P1B2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 238P1B2 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 238P1B2-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 238P1B2 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 238P1B2-related proteins that contain the amino acid residues of one or more of the biological motifs in a 238P1B2 protein are used to screen for factors that interact with that region of 238P1B2.

238P1B2 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 238P1B2 protein), for identifying agents or cellular factors that bind to 238P1B2 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 238P1B2 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to an 238P1B2 gene product. Antibodies raised against an 238P1B2 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 238P1B2 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 238P1B2-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 238P1B2 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 238P1B2-expressing cells (e.g., in radioscintigraphic imaging methods). 238P1B2 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 238P1B2 Antibodies

Another aspect of the invention provides antibodies that bind to 238P1B2-related proteins. Preferred antibodies specifically bind to a 238P1B2-related protein and do not bind (or bind weakly) to peptides or proteins that are not 238P1B2-related proteins. For example, antibodies that bind 238P1B2 can bind 238P1B2-related proteins such as the homologs or analogs thereof.

238P1B2 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 238P1B2 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 238P1B2 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 238P1B2 and mutant 238P1B2-related proteins. Such assays can comprise one or more 238P1B2 antibodies capable of recognizing and binding a 238P1B2-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 238P1B2 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 238P1B2 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 238P1B2 expressing cancers such as prostate cancer.

238P1B2 antibodies are also used in methods for purifying a 238P1B2-related protein and for isolating 238P1B2 homologues and related molecules. For example, a method of purifying a 238P1B2-related protein comprises incubating an 238P1B2 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 238P1B2-related protein under conditions that permit the 238P1B2 antibody to bind to the 238P1B2-related protein; washing the solid matrix to eliminate impurities; and eluting the 238P1B2-related protein from the coupled antibody. Other uses of 238P1B2 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 238P1B2 protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 238P1B2-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 238P1B2 can also be used, such as a 238P1B2 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 238P1B2-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 238P1B2-related protein or 238P1B2 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 238P1B2 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 238P1B2 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 238P1B2 amino acid sequence are used to identify hydrophilic regions in the 238P1B2 structure. Regions of a 238P1B2 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 238P1B2 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 238P1B2 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

238P1B2 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 238P1B2-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 238P1B2 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 238P1B2 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 238P1B2 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 238P1B2 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 238P1B2 antibodies with an 238P1B2-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 238P1B2-related proteins, 238P1B2-expressing cells or extracts thereof. A 238P1B2 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 238P1B2 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 238P1B2 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or, the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity, including:

1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al, Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including ⁵¹Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 238P1B2 Transgenic Animals

Nucleic acids that encode a 238P1B2-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 238P1B2 can be used to clone genomic DNA that encodes 238P1B2. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 238P1B2. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 238P1B2 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 238P1B2 can be used to examine the effect of increased expression of DNA that encodes 238P1B2. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 238P1B2 can be used to construct a 238P1B2 “knock out” animal that has a defective or altered gene encoding 238P1B2 as a result of homologous recombination between the endogenous gene encoding 238P1B2 and altered genomic DNA encoding 238P1B2 introduced into an embryonic cell of the animal. For example, cDNA that encodes 238P1B2 can be used to clone genomic DNA encoding 238P1B2 in accordance with established techniques. A portion of the genomic DNA encoding 238P1B2 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 238P1B2 polypeptide.

VII.) Methods for the Detection of 238P1B2

Another aspect of the present invention relates to methods for detecting 238P1B2 polynucleotides and 238P1B2-related proteins, as well as methods for identifying a cell that expresses 238P1B2. The expression profile of 238P1B2 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 238P1B2 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 238P1B2 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of 238P1B2 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 238P1B2 polynucleotides include, for example, a 238P1B2 gene or fragment thereof, 238P1B2 mRNA, alternative splice variant 238P1B2 mRNAs, and recombinant DNA or RNA molecules that contain a 238P1B2 polynucleotide. A number of methods for amplifying and/or detecting the presence of 238P1B2 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 238P1B2 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an 238P1B2 polynucleotides as sense and antisense primers to amplify 238P1B2 cDNAs therein; and detecting the presence of the amplified 238P1B2 cDNA. Optionally, the sequence of the amplified 238P1B2 cDNA can be determined.

In another embodiment, a method of detecting a 238P1B2 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 238P1B2 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 238P1B2 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 238P1B2 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an 238P1B2 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 238P1B2-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 238P1B2-related protein in a biological sample comprises first contacting the sample with a 238P1B2 antibody, a 238P1B2-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 238P1B2 antibody; and then detecting the binding of 238P1B2-related protein in the sample.

Methods for identifying a cell that expresses 238P1B2 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 238P1B2 gene comprises detecting the presence of 238P1B2 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 238P1B2 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 238P1B2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 238P1B2 gene comprises detecting the presence of 238P1B2-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 238P1B2-related proteins and cells that express 238P1B2-related proteins.

238P1B2 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 238P1B2 gene expression. For example, 238P1B2 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 238P1B2 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 238P1B2 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 238P1B2-related Genes and Their Products

Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 238P1B2 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 238P1B2 in a biological sample of interest can be compared, for example, to the status of 238P1B2 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 238P1B2 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 238P1B2 status in a sample.

The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 238P1B2 expressing cells) as well as the level, and biological activity of expressed gene products (such as 238P1B2 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 238P1B2 comprises a change in the location of 238P1B2 and/or 238P1B2 expressing cells and/or an increase in 238P1B2 mRNA and/or protein expression.

238P1B2 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 238P1B2 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 238P1B2 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 238P1B2 gene), Northern analysis and/or PCR analysis of 238P1B2 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 238P1B2 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 238P1B2 proteins and/or associations of 238P1B2 proteins with polypeptide binding partners). Detectable 238P1B2 polynucleotides include, for example, a 238P1B2 gene or fragment thereof, 238P1B2 mRNA, alternative splice variants, 238P1B2 mRNAs, and recombinant DNA or RNA molecules containing a 238P1B2 polynucleotide.

The expression profile of 238P1B2 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 238P1B2 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 238P1B2 status and diagnosing cancers that express 238P1B2, such as cancers of the tissues listed in Table I. For example, because 238P1B2 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 238P1B2 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 238P1B2 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 238P1B2 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 238P1B2 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 238P1B2 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 238P1B2 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 238P1B2 expressing cells (e.g. those that express 238P1B2 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 238P1B2-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 238P1B2 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 238P1B2 gene products by determining the status of 238P1B2 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 238P1B2 gene products in a corresponding normal sample. The presence of aberrant 238P1B2 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 238P1B2 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 238P1B2 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 238P1B2 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 238P1B2 mRNA or express it at lower levels.

In a related embodiment, 238P1B2 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 238P1B2 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 238P1B2 expressed in a corresponding normal sample. In one embodiment, the presence of 238P1B2 protein is evaluated, for example, using immunohistochemical methods. 238P1B2 antibodies or binding partners capable of detecting 238P1B2 protein expression are used in a variety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 238P1B2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 238P1B2 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 238P1B2 indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 238P1B2 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 238P1B2 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-1 tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of 238P1B2. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 238P1B2 expression. The presence of RT-PCR amplifiable 238P1B2 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 238P1B2 mRNA or 238P1B2 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 238P1B2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 238P1B2 in prostate or other tissue is examined, with the presence of 238P1B2 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 238P1B2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 238P1B2 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 238P1B2 mRNA or 238P1B2 protein expressed by tumor cells, comparing the level so determined to the level of 238P1B2 mRNA or 238P1B2 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 238P1B2 mRNA or 238P1B2 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 238P1B2 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 238P1B2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 238P1B2 mRNA or 238P1B2 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 238P1B2 mRNA or 238P1B2 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 238P1B2 mRNA or 238P1B2 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 238P1B2 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 238P1B2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 238P1B2 gene and 238P1B2 gene products (or perturbations in 238P1B2 gene and 238P1B2 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 238P1B2 gene and 238P1B2 gene products (or perturbations in 238P1B2 gene and 238P1B2 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between the expression of 238P1B2 gene and 238P1B2 gene products (or perturbations in 238P1B2 gene and 238P1B2 gene products) and another factor associated with malignancy entails detecting the overexpression of 238P1B2 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 238P1B2 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 238P1B2 and PSA mRNA in prostate tissue is examined, where the coincidence of 238P1B2 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 238P1B2 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 238P1B2 mRNA include in situ hybridization using labeled 238P1B2 riboprobes, Northern blot and related techniques using 238P1B2 polynucleotide probes, RT-PCR analysis using primers specific for 238P1B2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 238P1B2 mRNA expression. Any number of primers capable of amplifying 238P1B2 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 238P1B2 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 238P1B2

The 238P1B2 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 238P1B2, as well as pathways activated by 238P1B2 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 238P1B2 protein sequences. In such methods, peptides that bind to 238P1B2 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 238P1B2 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 238P1B2 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 238P1B2 are used to identify protein-protein interactions mediated by 238P1B2. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 238P1B2 protein can be immunoprecipitated from 238P1B2-expressing cell lines using anti-238P1B2 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 238P1B2 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, ³⁵S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 238P1B2 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 238P1B2's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 238P1B2-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 238P1B2 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 238P1B2 function can be identified based on their ability to bind 238P1B2 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 238P1B2 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators which activate or inhibit 238P1B2.

An embodiment of this invention comprises a method of screening for a molecule that interacts with an 238P1B2 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 238P1B2 amino acid sequence, allowing the population of molecules and the 238P1B2 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 238P1B2 amino acid sequence, and then separating molecules that do not interact with the 238P1B2 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 238P1B2 amino acid sequence. The identified molecule can be used to modulate a function performed by 238P1B2. In a preferred embodiment, the 238P1B2 amino acid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 238P1B2 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 238P1B2 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a 238P1B2 protein are useful for patients suffering from a cancer that expresses 238P1B2. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 238P1B2 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 238P1B2 gene or translation of 238P1B2 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 238P1B2-related protein or 238P1B2-related nucleic acid. In view of the expression of 238P1B2, cancer vaccines prevent and/or treat 238P1B2-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 238P1B2-related protein, or an 238P1B2-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 238P1B2 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 238P1B2 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 238P1B2 immunogen contains a biological motif, see e.g., Tables V-XVIII, Table XIX, or a peptide of a size range from 238P1B2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 238P1B2 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.

In patients with 238P1B2-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within 238P1B2 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimemm and Epimatrix™, Brown University; and, BIMAS, and SYFPEITHI). In a preferred embodiment, a 238P1B2 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII, Table XIX, or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-based Vaccines

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 238P1B2 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 238P1B2 in a host, by contacting the host with a sufficient amount of at least one 238P1B2 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 238P1B2 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 238P1B2-related protein or a man-made multiepitopic peptide comprising: administering 238P1B2 immunogen (e.g. a 238P1B2 protein or a peptide fragment thereof, an 238P1B2 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 238P1B2 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 238P1B2 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 238P1B2, in order to generate a response to the target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 238P1B2. Constructs comprising DNA encoding a 238P1B2-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 238P1B2 protein/immunogen. Alternatively, a vaccine comprises a 238P1B2-related protein. Expression of the 238P1B2-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 238P1B2 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at the world wide web Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 238P1B2-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Thus, gene delivery systems are used to deliver a 238P1B2-related nucleic acid molecule. In one embodiment, the full-length human 238P1B2 cDNA is employed. In another embodiment, 238P1B2 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 238P1B2 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 238P1B2 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 238P1B2 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 238P1B2 protein. Yet another embodiment involves engineering the overexpression of a 238P1B2 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 238P1B2 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

X.B.) 238P1B2 as a Target for Antibody-based Therapy

238P1B2 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 238P1B2 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 238P1B2-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 238P1B2 are useful to treat 238P1B2-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

238P1B2 antibodies can be introduced into a patient such that the antibody binds to 238P1B2 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 238P1B2, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 238P1B2 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 238P1B2), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-238P1B2 antibody) that binds to a marker (e.g. 238P1B2) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 238P1B2, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 238P1B2 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-238P1B2 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al, 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 238P1B2 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄ kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 238P1B2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Although 238P1B2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 238P1B2 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 238P1B2 imaging, or other techniques that reliably indicate the presence and degree of 238P1B2 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

Anti-238P1B2 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-238P1B2 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-238P1B2 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 238P1B2. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-238P1B2 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 238P1B2 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-238P1B2 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-238P1B2 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-238P1B2 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-238P1B2 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-238P1B2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-238P1B2 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 238P1B2 expression in the patient, the extent of circulating shed 238P1B2 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 238P1B2 in a given sample (e.g. the levels of circulating 238P1B2 antigen and/or 238P1B2 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-238P1B2 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 238P1B2-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-238P1B2 antibodies that mimic an epitope on a 238P1B2-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

X.C.) 238P1B2 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 238P1B2 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142). A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 238P1B2, the PADRE® universal helper T cell epitope (or multiple HTL epitopes from 238P1B2), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by ⁵¹Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.

For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID: 1), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID: 2), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID: 3). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID: 4), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P₃CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 238P1B2. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 238P1B2.

X.D. Adoptive Immunotherapy

Antigenic 238P1B2-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 238P1B2. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 238P1B2. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 238P1B2-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 238P1B2, a vaccine comprising 238P1B2-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.

Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administration of the anti-238P1B2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-238P1B2 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 238P1B2 expression in the patient, the extent of circulating shed 238P1B2 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body area weekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 10⁴ cells to about 10⁶ cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹ cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶ cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸ cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 238P1B2.

As disclosed herein, 238P1B2 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in Example 4).

238P1B2 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of 238P1B2 polynucleotides and polypeptides (as well as 238P1B2 polynucleotide probes and anti-238P1B2 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 238P1B2 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 238P1B2 polynucleotides described herein can be utilized in the same way to detect 238P1B2 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 238P1B2 polypeptides described herein can be utilized to generate antibodies for use in detecting 238P1B2 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 238P1B2 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 238P1B2-expressing cells (lymph node) is found to contain 238P1B2-expressing cells such as the 238P1B2 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 238P1B2 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 238P1B2 or express 238P1B2 at a different level are found to express 238P1B2 or have an increased expression of 238P1B2 (see, e.g., the 238P1B2 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 238P1B2) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 238P1B2 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in Example 4, where a 238P1B2 polynucleotide fragment is used as a probe to show the expression of 238P1B2 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov.-Dec. 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 238P1B2 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 238P1B2 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 238P1B2 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 238P1B2 polypeptide shown in FIG. 3).

As shown herein, the 238P1B2 polynucleotides and polypeptides (as well as the 238P1B2 polynucleotide probes and anti-238P1B2 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 238P1B2 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 238P1B2 polynucleotides and polypeptides (as well as the 238P1B2 polynucleotide probes and anti-238P1B2 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 238P1B2 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 238P1B2 gene maps (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the 238P1B2-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

Additionally, 238P1B2-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 238P1B2. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 238P1B2 antigen. Antibodies or other molecules that react with 238P1B2 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 238P1B2 Protein Function

The invention includes various methods and compositions for inhibiting the binding of 238P1B2 to its binding partner or its association with other protein(s) as well as methods for inhibiting 238P1B2 function.

XII.A.) Inhibition of 238P1B2 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 238P1B2 are introduced into 238P1B2 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-238P1B2 antibody is expressed intracellularly, binds to 238P1B2 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL (SEQ ID: 5) amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 238P1B2 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 238P1B2 intrabodies in order to achieve the desired targeting. Such 238P1B2 intrabodies are designed to bind specifically to a particular 238P1B2 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 238P1B2 protein are used to prevent 238P1B2 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 238P1B2 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 238P1B2 with Recombinant Proteins

In another approach, recombinant molecules bind to 238P1B2 and thereby inhibit 238P1B2 function. For example, these recombinant molecules prevent or inhibit 238P1B2 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 238P1B2 specific antibody molecule. In a particular embodiment, the 238P1B2 binding domain of a 238P1B2 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 238P1B2 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 238P1B2, whereby the dimeric fusion protein specifically binds to 238P1B2 and blocks 238P1B2 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of 238P1B2 Transcription or Translation

The present invention also comprises various methods and compositions for inhibiting the transcription of the 238P1B2 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 238P1B2 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 238P1B2 gene comprises contacting the 238P1B2 gene with a 238P1B2 antisense polynucleotide. In another approach, a method of inhibiting 238P1B2 mRNA translation comprises contacting a 238P1B2 mRNA with an antisense polynucleotide. In another approach, a 238P1B2 specific ribozyme is used to cleave a 238P1B2 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 238P1B2 gene, such as 238P1B2 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 238P1B2 gene transcription factor are used to inhibit 238P1B2 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 238P1B2 by interfering with 238P1B2 transcriptional activation are also useful to treat cancers expressing 238P1B2. Similarly, factors that interfere with 238P1B2 processing are useful to treat cancers that express 238P1B2. Cancer treatment methods utilizing such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 238P1B2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 238P1B2 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 238P1B2 antisense polynucleotides, ribozymes, factors capable of interfering with 238P1B2 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 238P1B2 to a binding partner, etc.

In vivo, the effect of a 238P1B2 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

XIII.) Kits

For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 238P1B2-related protein or a 238P1B2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 238P1B2 Gene

The Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from patient cancer tissues is used to isolate genes that are over-expressed in cancer. The 238P1B2 SSH cDNA sequence was derived from a colon cancer pool minus normal tissue cDNA subtraction. Included in the driver were cDNAs derived from 10 normal tissues. The 238P1B2 cDNA was identified as highly expressed in the prostate cancer tissue pool, with restricted expression detected in normal tissues.

The SSH DNA sequence of 210 bp (FIG. 1) is novel but shows homology to mouse olfactory receptor MOR14-10 mRNA. A 238P1B2 cDNA, 238P1B2-clone A, of 3754 bp was isolated from prostate cDNA library, revealing an ORF of 254 amino acids (FIG. 2, FIG. 3 and FIG. 4A).

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, Pa.). mRNA for some normal tissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID: 6) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQ ID: 7) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID: 8) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID: 9) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID: 10) 3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID: 11) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQ ID: 12) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID: 13) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that are differentially expressed in cancer. The SSH reaction utilized cDNA from colon cancer and normal tissues.

The gene 238P1B2 sequence was derived from a colon cancer pool minus normal tissue cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from of a pool of normal tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of patient cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:

To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer I (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID: 14) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID: 15) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 238P1B2 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 238P1B2 SSH sequence and are listed below:

238P1B2.1 5′- TTGCAGAATATCACCTCCACTTCC -3′ (SEQ ID: 16) 238P1B2.2 5′- GATCAGGCTGTTTCCCAAGAGAG -3′ (SEQ ID: 17)

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Results show strong expression of 238P1B2 in prostate cancer pool but not in vital pool 1 and vital pool 2.

Example 2 Full Length Cloning of 238P1B2

The 238P1B2 SSH cDNA sequence was derived from a colon cancer pool minus normal tissue cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 238P1B2.

The SSH DNA sequence of 210 bp (FIG. 1) is novel and shows 91% identity to mouse olfactory receptor MOR14-10 mRNA.

A full length cDNA (238P1B2-clone A) of 3754 bp was isolated from prostate library, revealing an ORF of 254 amino acids (FIGS. 2 and 3). The cDNA shows highest homology to the mouse MOR-14-1 and MOR14-10 olfactory receptors, with identities of 85% over 912 nucleotides and 83% over 906 nucleotides respectively (FIG. 4).

The protein sequence reveals 7 transmembrane domains and has homology to G protein-coupled receptors (GPCRs) involved in olfaction (Raming et al., 1993, Nature 361: 353; Malnic et al., 1999, Cell 96:713). Proteins that are members of this receptor family exhibit an extracellular amino-terminus, three additional extracellular loops, three intracellular loops and an intracellular carboxyl terminus.

The most homologous sequence to 238P1B2 is mouse MOR14-1 protein. The two proteins share 83% identity over a 253 amino acid region. Alignment of the two proteins is shown in FIG. 4.

238P1B2 also shows significant homology to the prostate specific GPCR, PHOR-1. The two proteins share 48% identity over a region of 250 amino acids (FIG. 4).

The full length 238P1B2 cDNA (238P1B2-clone A) was deposited with the American Type Culture Collection on Mar. 7, 2002, and assigned accession number PTA-4124.

238P1B2 v.1A protein sequence codes for a six transmembrane protein. Extension of the protein at the amino terminus adds another 62 amino acids to 238P1B2 v.1A leading to 238P1B2 v.1B. This 62 amino acid terminus of 238P1B2 v.1B encodes an additional transmembrane region, and thereby making 238P1B2 v.1B a seven transmembrane protein. Furthermore, this amino terminus shows 84% identity to the mouse MOR14-10 G-coupled protein receptor, indicating that this amino-terminal portion is encoded in nature.

The natural expression of 238P1B2 v.1B can occur if the stop site upstream of the 62 amino acid extension is modified by single nucleic acid substitution leading to conversion of the stop codon into a coding amino acid. Polymorphisms can exist within different tissues, or between different individuals that mutate the stop codon, and therefore allowing proper translation of the 62 amino acid amino-terminal region of 238P1B2 v.1B. Such a variation can either add a start methionine directly, or extend the coding region 238P1B2 v.1B for an additional 16 amino acids until reaching a start methionine at nucleic acid position 1896.

Example 3 Chromosomal Mapping of 238P1B2 Gene

Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Alabama), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).

Using 238P1B2 sequence and the NCBI BLAST tool, placed 238P1B2 to chromosome 11p15.5, a region rich in GPCRs.

Because the human 238P1B2 gene maps to chromosome 11p15.5, polynucleotides encoding different regions of the 238P1B2 protein can be used to characterize cytogenetic abnormalities on chromosome 11, band p15.5 that have been identified as being associated with various cancers. In particular, a variety of chromosomal abnormalities in 11p15.5 have been identified as frequent cytogenetic abnormalities in a number of different cancers (see, e.g., Lai et al., 2000, Clin. Cancer Res. 6(8):3172-6; Oya and Schulz, 2000, Br. J. Cancer 83(5):626-31; Svaren et al., Sep. 12, 2000, J. Biol. Chem.). Consequently, polynucleotides encoding specific regions of the 238P1B2 protein provide new tools that can be used to delineate, with greater precision than previously possible, the specific nature of the cytogenetic abnormalities in this region of chromosome 11 that contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see, e.g., Evans et al., 1994, Am. J. Obstet. Gynecol. 171(4):1055-1057).

Example 4 Expression Analysis of 238P1B2 in Normal Tissues and Patient Specimens

Expression analysis by RT-PCR demonstrated that 238P1B2 is strongly expressed in prostate cancer patient specimens (FIG. 14). First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach) and prostate cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 238P1B2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 238P1B2 in prostate cancer pool but not in vital pool 1 and vital pool 2.

Northern blot analysis of 238P1B2 in 16 human normal tissues is shown in FIG. 15. Results show absence of 238P1B2 expression in all 16 normal tissues tested. Extensive analysis of expression of 238P1B2 in 76 human tissues shows restricted expression of 238P1B2 in placenta (FIG. 16).

Expression of 238P1B2 in patient cancer specimens and human normal tissues is shown in FIG. 17. RNA was extracted from a pool of three prostate cancers, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC), normal lung (NL) normal breast (NBr) and normal ovary (NO). Northern blot with 10 μg of total RNA/lane was probed with 238P1B2 sequence. The results show expression of an approximately 4.5 kb 238P1B2 transcript in the prostate cancer pool and ovary but not in the other normal tissues tested. Analysis of individual patient specimens shows strong expression of 238P1B2 in prostate cancer tissues (FIG. 18). The expression for 238P1B2 detected in tumors of Gleason score 7 is significantly stronger than the expression detected in tumors of Gleason score 5. This result indicates that 238P1B2 can be used as a prognostic marker for prostate cancer.

The restricted expression of 238P1B2 in normal tissues and the expression detected in prostate cancer suggest that 238P1B2 is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 238P1B2

Transcript variants are variants of mature mRNA from the same gene by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene that start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiments, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail and GenScan. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 238P1B2 has a particular expression profile. Alternative transcripts and splice variants of 238P1B2 can share this expression pattern, thus serving as tumor-associated markers/antigens.

The exon composition of the original transcript, designated as 238P1B2 v.1, is shown in Table XXIII. No transcript variant has been identified by the above methods.

Example 6 Single Nucleotide Polymorphisms of 238P1B2

A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1): 15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).

238P1B2 SNPs are identified by direct sequencing of the cDNA clones and by comparison of those sequences with public and proprietary sequences. By comparing the sequences with high quality proprietary or public sequences (e.g., NCBI/GenBank, accessible at World Wide Web URL ncbi.nlm.nih.gov), five SNPs of 238P1B2 were identified at nucleotide positions 274 (T/C), 1268 (T/G), 1299 (T/G) 2806 (T/C) and 3025 (T/C). The transcripts or proteins with alternative alleles were designated as variants 238P1B2 v.2, v.3, v.4, v.5 and v.6. FIG. 10 shows the schematic alignment of the nucleotide variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in FIG. 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes).

Example 7 Production of Recombinant 238P1B2 in Prokaryotic Systems

To express recombinant 238P1B2 in prokaryotic cells, the full or partial length 238P1B2 variant 1a, variant 1b, and variant 2 cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 238P1B2 variants are expressed in these constructs: amino acids 1 to 254 of variant 1a; amino acids 1 to 316 of variant 1b, and amino acids 1-254 of variant 2, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous amino acids from 238P1B2, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 238P1B2 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all of or fragments of the 238P1B2 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 238P1B2 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 238P1B2 at the RNA level. Transcribed 238P1B2 RNA representing the cDNA amino acid coding region of the 238P1B2 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 238P1B2 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 238P1B2 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all of or parts of the 238P1B2 cDNA protein coding sequence are fused to the GST gene by cloning into pGEX-6P-1 or any other GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 238P1B2 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus. The GST and 6×His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6×His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, can be employed to permit cleavage of the GST tag from 238P1B2-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 238P1B2 proteins that are fused to maltose-binding protein (MBP), all of or parts of the 238P1B2 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 238P1B2 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6×His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 238P1B2. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs: To express 238P1B2 in bacterial cells, all of or parts of the 238P1B2 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 238P1B2 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 238P1B2 protein are expressed as amino-terminal fusions to NusA. In one embodiment, a NusA-fusion protein encompassing amino acids 412-254 of 238P1B2 with a C-terminal 6×His tag was expressed in E. Coli, purified by metal chelate affinity chromatography, and used as an immunogen for generation of antibodies.

C. Yeast Constructs:

pESC Constructs: To express 238P1B2 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all of or parts of the 238P1B2 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 238P1B2. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 238P1B2 in the yeast species Saccharomyces pombe, all of or parts of the 238P1B2 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 238P1B2 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 238P1B2 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 238P1B2 in eukaryotic cells, the full or partial length 238P1B2 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 238P1B2 are expressed in these constructs: amino acids 1 to 254 of variant 1A or variant 2, amino acids 1 to 316 of variant 1B, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous amino acids from 238P1B2, variants, or analogs thereof. In certain embodiments a region of a specific variant of 238P1B2 is expressed that encodes an amino acid at a specific position which differs from the amino acid of any other variant found at that position. In other embodiments, a region of a variant of 238P1B2 is expressed that lies partly or entirely within a sequence that is unique to that variant.

The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-238P1B2 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 238P1B2 in mammalian cells, a 238P1B2 ORF, or portions thereof, of 238P1B2 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6×His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 238P1B2 in mammalian cells, a 238P1B2 ORF, or portions thereof, of 238P1B2 with a consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 238P1B2 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 238P1B2 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 238P1B2 protein.

PAPtag: A 238P1B2 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 238P1B2 protein while fusing the IgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminus of a 238P1B2 protein. The resulting recombinant 238P1B2 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 238P1B2 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

ptag5: A 238P1B2 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 238P1B2 protein with an amino-terminal IgGκ signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 238P1B2 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 238P1B2 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: A 238P1B2 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 238P1B2 proteins, while fusing the IgGK signal sequence to N-terminus. 238P1B2 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 238P1B2 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 238P1B2 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 238P1B2 constitutively, 238P1B2 ORF, or portions thereof, of 238P1B2 are cloned into pSRα constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 238P1B2, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 238P1B2 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID: 18) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 238P1B2 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 238P1B2. High virus titer leading to high level expression of 238P1B2 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 238P1B2 coding sequence or fragments thereof is amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 238P1B2 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 238P1B2 in mammalian cells, coding sequences of 238P1B2, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 238P1B2. These vectors are thereafter used to control expression of 238P1B2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 238P1B2 proteins in a baculovirus expression system, 238P1B2 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-238P1B2 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 238P1B2 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 238P1B2 protein can be detected using anti-238P1B2 or anti-His-tag antibody. 238P1B2 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 238P1B2.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5A,B, FIG. 6A,B, FIG. 7A,B, FIG. 8A,B, and FIG. 9A,B depict graphically five amino acid profiles of the 238P1B2 variant 1a (5A-9a) amino acid sequence and variant 1b (5B-9B), each assessment available by accessing the ProtScale website on the ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 238P1B2 protein. Each of the above amino acid profiles of 238P1B2 were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus be available for immune recognition, such as by antibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible for immune recognition, such as by antibodies.

Antigenic sequences of the 238P1B2 variant 1a and variant 1b protein indicated, e.g., by the profiles set forth in FIG. 5A,B, FIG. 6 A,B, FIG. 7A,B, FIG. 8A,B, and/or FIG. 9A,B are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-238PIB2 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 238P1B2 variant proteins. In particular, peptide immunogens for 238P1B2 variant 1a of the invention can comprise, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 254 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5A; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 254 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6A; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 254 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7A; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 254 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8A; and, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 254 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9A.

In addition, peptide immunogens for 238P1B2 variant 1b of the invention can comprise, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 316 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5B; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 316 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6B; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 316 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7B; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 316 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8B; and, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 316 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9B. Immunogens of the invention can also comprise nucleic acids that encode any of the foregoing peptides.

All immunogens of the invention, whether peptides or nucleic acids, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 238P1B2, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (Guermeur, 1997), accessed from the ExPasy molecular biology server. The analysis indicates that 238P1B2 variant 1a is composed of 66.54% alpha helix, 6.69% extended strand, and 26.77% random coil (FIG. 12A). The analysis indicates that 238P1B2 variant 1b is composed of 61.71% alpha helix, 8.86% extended strand, and 29.43% random coil (FIG. 12B).

Analysis for the potential presence of transmembrane domains in 238P1B2 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. The programs predict the presence of multiple transmembrane domains in 238P1B2 variant 1a and variant 1b. The highest probability of topology for variant 1a is that of a cell surface protein with 6 transmembrane domains. The highest probability of topology for variant 1b is that of a cell surface protein with 7 transmembrane domains, consistent with that of a G-protein coupled receptor. Shown graphically in FIG. 12C and FIG. 12D are the results of analysis of 238P1B2 variant 1a using the TMpred (FIG. 12C) and TMHMM (FIG. 12D) prediction programs depicting the location and topology of the 6 transmembrane domains. Shown in FIG. 12E and FIG. 12F are the results of the prediction programs for 238P1B2 variant 1b showing the location and topology of the 7 transmembrane domains. The results of each program, namely the amino acids encoding the transmembrane domain, are summarized in Table XXII.

Example 10 Generation of 238P1B2 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 238P1B2 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and/or be exposed on the surface of the protein (see, e.g., FIG. 5A,B, FIG. 6A,B, FIG. 7A,B, FIG. 8A,B, and FIG. 9A,B for amino acid profiles that indicate such regions of 238P1B2 variants 1a and 1b).

For example, 238P1B2 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of the 238P1B2 variant 1, generally found in regions between transmembrane domains and at the amino and carboxyl termini, are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. Examples of such regions include, but are not limited to, amino acids 30-43, amino acids 109-141, and amino acids 208-211, which are regions that are predicted to be extracellular; and amino acids 67-85, amino acids 165-184, and amino acids 235-254, which are regions predicted to be intracellular. In addition, the amino-terminal region of variant 1b, amino acids 1-29 can be used as an immunogen. Antibodies to this region are useful to distinguish variant 1b protein from variant 1a protein. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 1-29 of 238P1B2 variant 1b is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent can include all or portions of the 238P1B2 protein, analogs or fusion proteins thereof. For example, the 238P1B2 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding amino acids 1-254 of variant 1a is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that can be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 238P1B2 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 238P1B2 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 109-141 of variant 1a are cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 238P1B2 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with Tag5 238PIB2 variant 1a encoding amino acids 109-141, the full-length 238P1B2 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 238P1B2 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-238P1B2 antibodies and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity of the antibodies to denatured 238P1B2 protein using the Western blot technique. Immunoprecipitation and flow cytometric analyses of 293T and other recombinant 238P1B2-expressing cells determine recognition of native protein by the antibodies. In addition, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 238P1B2 are carried out to test specificity.

Anti-serum from rabbits immunized with 238P1B2 fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-238P1B2 fusion protein encoding amino acids 1-254 of variant 1a is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-fusion protein also encoding amino acids 1-254 of variant 1a covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11 Generation of 238P1B2 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 238P1B2 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 238P1B2, for example antibodies that disrupt its interaction with ligands and binding partners. Therapeutic mAbs also comprise those that specifically bind epitopes of 238P1B2 exposed on the cell surface and thus are useful in targeting mAb-toxin conjugates. Immunogens for generation of such mAbs include those designed to encode or contain the entire 238P1B2 protein, regions of the 238P1B2 protein predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5A,B, FIG. 6A,B, FIG. 7A,B, FIG. 8A,B, or FIG. 9A,B, and the Example entitled “Antigenicity Profiles”), and regions such as predicted extracellular domains. Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells expressing high levels of 238P1B2, such as 293T-238P1B2 or 300.19-238P1B2 murine Pre-B cells, are used to immunize mice.

To generate mAbs to 238P1B2, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ 238P1B2-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding 238P1B2 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 109-141 is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 238P1B2 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing 238P1B2.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 238P1B2 monoclonal antibodies, a Tag5-238P1B2 antigen encoding amino acids 109-141 of variant 1a, a predicted extracellular domain, is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5 238P1B2 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 238P1B2 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 238P1B2 cDNA (see e.g., the Example entitled “Production of Recombinant 238P1B2 in Eukaryotic Systems”). Other recombinant 238P1B2-expressing cells or cells endogenously expressing 238P1B2 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 238P1B2 specific antibody-producing clones.

The binding affinity of a 238P1B2 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 238P1B2 monoclonal antibodies are suitable for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a useful method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measured IC₅₀ values are reasonable approximations of the true K_(D) values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC₅₀ of a positive control for inhibition by the IC₅₀ for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC₅₀ nM values by dividing the IC₅₀ nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides.

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/or Motif-bearing Epitopes

The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables V-XVIII and Table XIX, employ the protein sequence data from the gene product of 238P1B2 set forth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 238P1B2 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: “ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni) where a_(ji) is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount j_(i) to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of j_(i). For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 Supertype Cross-reactive Peptides

Protein sequences from 238P1B2 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.

Selection of HLA-A3 Supermotif-bearing Epitopes

The 238P1B2 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦500 nM, often ≦200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 238P1B2 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC₅₀ of ≦500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 238P1B2 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:

Target Cell Lines for Cellular Screening:

The 221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC are processed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×10⁶ cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵ cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶ cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a ⁵¹Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) ⁵¹Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 10⁶ per ml and diluted 1:10 with K562 cells at a concentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula: [(cpm of the test sample−cpm of the spontaneous ⁵¹Cr release sample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous ⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates are washed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at 400 μg or 1200 μg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H₃PO₄ and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flask containing the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV—transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×10⁶/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assay or at 1×10⁶/ml in the in situ IFNγ assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25 flask containing the following: 1×10⁶ autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

Immunogenicity of A2 Supermotif-bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 238P1B2. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus. To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of 5000 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

Analoging of HLA-A3 and B7-supermotif-bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to ⅗ of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position I is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 238P1B2-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g. the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.

Example 16 Identification and Confirmation of 238P1B2-derived Sequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-supermotif-bearing Epitopes.

To identify 238P1B2-derived, HLA class II HTL epitopes, a 238P1B2 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 238P1B2-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 238P1B2-derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 238P1B2 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 17 Immunogenicity of 238P1B2-derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 238P1B2-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.

Example 19 CTL Recognition Of Endogenously Processed Antigens After Priming

This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells in the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 238P1B2 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 238P1B2 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*020₁/K^(b) transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 20 Activity Of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 238P1B2-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 238P1B2-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/K^(b) mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) are incubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 10⁴ ⁵¹Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specific lytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptide is subtracted from the lytic units/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Cr release is obtained at the effector (E):

target (T) ratio of 50:1 (i.e., 5×10⁵ effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [( 1/50,000)−( 1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity”. Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in an 238P1B2-specific Vaccine

This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responses that are correlated with 238P1B2 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 238P1B2. For example, if it has been observed that patients who spontaneously clear 238P1B2 generate an immune response to at least three (3) from 238P1B2 antigen, then three or four (3-4) epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site.

In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 238P1B2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 238P1B2.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 238P1B2, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 238P1B2 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it Induces Immunogenicity

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a ⁵¹Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, 1-A^(b)-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent 238P1B2 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 238P1B2-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 238P1B2-associated disease.

Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 238P1B2 Sequences

A native 238P1B2 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes is selected; it can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 238P1B2 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 238P1B2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.

Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 238P1B2 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 238P1B2 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 238P1B2 as well as tumor-associated antigens that are often expressed with a target cancer associated with 238P1B2 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 238P1B2. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 238P1B2 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising an 238P1B2 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 238P1B2 epitope, and thus the status of exposure to 238P1B2, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 238P1B2-associated disease or who have been vaccinated with an 238P1B2 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 238P1B2 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release−spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 238P1B2 or an 238P1B2 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 238P1B2 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for ³H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of ³H-thymidine incorporation in the presence of antigen divided by the ³H-thymidine incorporation in the absence of antigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 238P1B2

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 238P1B2. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 238P1B2, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:

The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 238P1B2.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 238P1B2-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 238P1B2 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells (DC)

Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 238P1B2 protein from which the epitopes in the vaccine are derived.

For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×10⁶ DC per patient are typically administered, larger number of DC, such as 10⁷ or 10⁸ can also be provided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 10⁸ to 10¹⁰. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×10⁶ DC, then the patient will be injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 238P1B2 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 238P1B2. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 238P1B2 to isolate peptides corresponding to 238P1B2 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 238P1B2-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 238P1B2. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 238P1B2. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 238P1B2-encoding transcript.

Example 35 Purification of Naturally-occurring or Recombinant 238P1B2 Using 238P1B2 Specific Antibodies

Naturally occurring or recombinant 238P1B2 is substantially purified by immunoaffinity chromatography using antibodies specific for 238P1B2. An immunoaffinity column is constructed by covalently coupling anti-238P1B2 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 238P1B2 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 238P1B2 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/238P1B2 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 36 Identification of Molecules which Interact with 238P1B2

238P1B2, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 238P1B2, washed, and any wells with labeled 238P1B2 complex are assayed. Data obtained using different concentrations of 238P1B2 are used to calculate values for the number, affinity, and association of 238P1B2 with the candidate molecules.

Example 37 In Vivo Assay for 238P1B2 Tumor Growth Promotion

The effect of the 238P1B2 protein on tumor cell growth is evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously on each flank with 1×10⁶ of either PC3, DU145 or 3T3 cells containing tkNeo empty vector or 238P1B2. At least two strategies can be used: (1) Constitutive 238P1B2 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 238P1B2-expressing cells grow at a faster rate and whether tumors produced by 238P1B2-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cells orthotopically to determine if 238P1B2 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 238P1B2 inhibitory effect of candidate therapeutic compositions, such as for example, 238P1B2 intrabodies, 238P1B2 antisense molecules and ribozymes.

Example 38 238P1B2 Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo

The significant expression of 238P1B2 in prostate cancer tissues and its restrictive expression in normal tissues, together with its expected cell surface expression, makes 238P1B2 an excellent target for antibody therapy. Similarly, 238P1B2 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-238P1B2 mAbs in human prostate cancer xenograft mouse models is evaluated by using human cancer xenografts (Craft, N., et al., Cancer Res, 1999. 59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-238P1B2 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): p. 16-23).

Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic prostate cancer xenograft models and mouse kidney xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-238P1B2 mAbs inhibit formation of androgen-independent PC3-238P1B2 tumor xenografts. Anti-238P1B2 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-238P1B2 mAbs in the treatment of local and advanced stages of prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078).

Administration of the anti-238P1B2 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 238P1B2 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-238P1B2 mAbs for the treatment of local and metastatic prostate cancer. This example demonstrates that unconjugated 238P1B2 monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 238P1B2 mAbs

Materials and Methods

238P1B2 Monoclonal Antibodies:

Monoclonal antibodies are raised against 238P1B2 as described in the Example entitled “Generation of 238P1B2 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 238P1B2. Epitope mapping data for the anti-238P1B2 mAbs, as determined by ELISA and Western analysis, indicates that the antibodies recognize epitopes on the 238P1B2 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.

Cancer Xenografts and Cell Lines

Human cancer xenograft models as well as ICR-severe combined immunodeficient (SCID) mice injected with human cell lines expressing or lacking 238P1B2 are used to confirm the role of this gene in tumor growth and progression. Prostate xenograft tissue is passaged in 6- to 8-week-old male SCID mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra). The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS.

PC3-238P1B2 and 3T3-238P1B2 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-238P1B2 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cells, such as PC3, PC3-238P1B2, 3T3 or 3T3-238P1B2 cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-238P1B2 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078).

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. PC3 cells (5×10⁵) mixed with Matrigel are injected into each dorsal lobe in a 10-μl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. The mice are segregated into groups for the appropriate treatments, with anti-238P1B2 or control mAbs being injected i.p.

Anti-238P1B2 mAbs Inhibit Growth of 238P1B2-Expressing Xenograft-Cancer Tumors

The effect of anti-238P1B2 mAbs on tumor formation is tested by using PC3-238P1B2 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse prostate or kidney, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 μg, of anti-238P1B2 Ab, or b) PBS three times per week for two to five weeks.

A major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8).

Mice bearing established orthotopic 3T3-238P1B2 and PC3-238P1B2 tumors are administered 1000 μg injections of either anti-238P1B2 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-238P1B2 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-238P1B2 antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors, retard the growth of already established tumors, and prolong the survival of treated mice. Moreover, anti-238P1B2 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-238P1B2 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-238P1B2 Antibodies in Humans

Anti-238P1B2 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-238P1B2 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 238P1B2 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-238P1B2 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-238P1B2 mAb specifically binds to carcinoma cells. Thus, anti-238P1B2 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 238P1B2. Shedding or release of an extracellular domain of 238P1B2 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 238P1B2 by anti-238P1B2 antibodies in serum and/or urine samples from suspect patients.

Anti-238P1B2 antibodies that specifically bind 238P1B2 are used in therapeutic applications for the treatment of cancers that express 238P1B2. Anti-238P1B2 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-238P1B2 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “Monoclonal Antibody-mediated Inhibition of Prostate Tumors In vivo”). Conjugated and unconjugated anti-238P1B2 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in the following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-238P1B2 Antibodies In vivo

Antibodies are used in accordance with the present invention which recognize an epitope on 238P1B2, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 238P1B2 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-238P1B2 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-238P1B2 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-238P1B2 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

II.) Monotherapy: In connection with the use of the anti-238P1B2 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I¹³¹, Y⁹⁰) to anti-238P1B2 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 238P1B2. In connection with the use of the anti-238P1B2 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (¹¹¹In)-238P1B2 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 238P1B2 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-238P1B2 antibodies can be administered with doses in the range of 5 to 400 mg/m², with the lower doses used, e.g., in connection with safety studies. The affinity of anti-238P1B2 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-238P1B2 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-238P1B2 antibodies can be lower, perhaps in the range of 50 to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-238P1B2 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-238P1B2 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-238P1B2 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 238P1B2 expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 238P1B2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-238P1B2 antibodies are found to be safe upon human administration.

Example 41 Human Clinical Trial Adjunctive Therapy with Human Anti-238P1B2 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-238P1B2 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-238P1B2 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-238P1B2 antibody with dosage of antibody escalating from approximately about 25 mg/m² to about 275 mg/m² over the course of the treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 mg/m² 75 mg/m² 125 mg/m² 175 mg/m² 225 mg/m² 275 mg/m² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 238P1B2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-238P1B2 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-238P1B2 Antibody

Anti-238P1B2 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-238P1B2 antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-238P1B2 Antibody

Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-238P1B2 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 238P1B2 to Known Sequences

The 238P1B2 gene is homologous to a cloned and sequenced gene, namely the mouse olfactory receptor MOR14-1 (gi 18479244) (Zhang X, Firestein S. Nat Neurosci. 2002, 5:124), showing 83% identity and 90% homology to that gene product (FIG. 4A; FIG. 4C). The 238P1B2 protein shows 78% identity and 87% homology to another mouse olfactory receptor, namely MOR14-10 (gi 18480766). The closest human homolog to 238P1B2 is the human olfactory receptor 5BETA12 (51I2, gi 17456801), with 61% identity and 79% homology (FIG. 4E). Comparison of 238P1B2 to another member of the human olfactory receptor family, namely 101P3A11 (reference AGS patent), the 238P1B2 protein shows 48% identity and 70% homology to 101P3A11, with the first amino acid of 238P1B2 aligning with aa 62 of 101P3A11. The 238P1B2 variant 1A protein consists of 254 amino acids, with calculated molecular weight of 28.5 kDa, and pI of 9.2. 238P1B2 is a cell surface protein with some localization to the mitochondria and endoplasmic reticulum. Three forms of the 238P1B2 protein have been identified, with variant 238P1B2 V2 containing a isoleucine to threonine point mutation at amino acid 225, and variant 238P1B2 V1B containing an additional 62 aa at its amino-terminus (FIG. 4F). While 238P1B2 V1A is projected to have 6 transmembrane domains, 238P1B2 V1B contains 7 transmembrane domains, with the N-terminus oriented extracellularly and the C-terminus being intracellular (Table XXII, FIG. 13).

Motif analysis revealed the presence of several known motifs, including a 7 transmembrane olfactory receptor GPCR motif and a fibronectin Type III repeat. Proteins that are members of the G-protein coupled receptor family exhibit an extracellular amino-terminus, three extracellular loops, three intracellular loops and an intracellular carboxyl terminus. G-protein coupled receptors are seven-transmembrane receptors that are stimulated by polypeptide hormones, neurotransmitters, chemokines and phospholipids (Civelli O et al, Trends Neurosci. 2001, 24:230; Vrecl M et al., Mol Endocrinol. 1998, 12:1818). Ligand binding traditionally occurs between the first and second extracellular loops of the GPCR. Upon ligand binding GPCRs transduce signals across the cell surface membrane by associating with trimeric G proteins. Their signals are transmitted via trimeric guanine-nucleotide binding proteins (G proteins) to cell surface receptor, effector enzymes or ion channels (Simon et al., 1991, Science 252: 802). Signal transduction and biological output mediated by GPCR can be modulated through various mechanisms including peptide mimics, small molecule inhibitors and GPCR kinases or GRK (Pitcher J A et al, J Biol Chem. 1999, 3; 274:34531; Fawzi A B, et al. 2001, Mol. Pharmacol., 59:30).

Recently, GPCRs have also been shown to link to mitogenic signaling pathways of tyrosine kinases (Luttrell et al., 1999, Science 283: 655; Luttrell et al., 1999 Curr Opin Cell Biol 11: 177). GPCRs are regulated by phosphorylation mediated by GPCR kinases (GRKs), which themselves are indirectly activated by the GPCRs (Pitcher et al., 1998, Ann. Rev. Biochem. 67: 653). Olfactory GPCRs transmit their signals by activating the cAMP pathway via adenylate cyclase resulting in downstream signaling to protein kinase A, and by activating the phospholipase C pathway by generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Breer, 1993, Ciba Found Symp 179: 97; Bruch, 1996, Comp Biochem Physiol B Biochem Mol Biol 113:451). IP3 results in an increase in intracellular calcium, while DAG activates protein kinase C.

Recent studies have associated GPCRs with cellular transformation. In particular, KSHV G protein-coupled receptor was found to transform NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV-GPCR-transgenic mice (Schwarz M, Murphy P M. J Immunol 2001, 167:505). KSHV-GPCR was capable of producing its effect on endothelial cells and fibroblasts by activating defined signaling pathways, including the AKT survival pathway (Montaner S et al, Cancer Res 2001, 61:2641). In addition, KSHV-GPCR induced the activation of mitogenic pathways such as AP-1 and NFkB, resulting in the expression of pro-inflammatory genes (Schwarz M, Murphy P M. J Immunol 2001, 167:505). Other GPCRs associated with tumor formation include G2A, and PAR-1, which has been found to induce transformation of NIH 3T3 cells (Whitehead IP et al, Oncogene 2001, 20:1547).

Fibronectin repeat regions are motifs that mediate binding to a variety of substances such as heparin, collagen, fibrin and fibronectin receptors on cell surfaces. Due to their binding capacity fibronectins are involved in cell adhesion, cell differentiation, spreading, migration, and tumor metastasis (Nykvist P et al, J Biol Chem 2001, 276:38673; Danen E H, Yamada K M. J Cell Physiol 2001, 189:1; Nabeshima K et al, Histol Histopathol 1999, 14:1183). In addition, fibronectin enhances angiogenesis and de novo blood vessel formation by regulating the migration of endothelial cells, which constitute essential components of blood vessels (Urbich C et al, Arterioscler Thromb Vasc Biol 2002, 22:69), thereby enhancing tumor growth and survival.

This information indicates that 238P1B2 plays a role in the transformation of mammalian cells, induces mitogenic responses including activation of various signaling pathways, and regulates gene transcription by transmitting cell surface signals to the nucleus. Accordingly, when 238P1B2 functions as a regulator of cell transformation, tumor formation, or as a modulator of transcription involved in activating genes associated with inflammation, tumorigenesis or proliferation, 238P1B2 is used for therapeutic, diagnostic, prognostic and/or preventative purposes. In addition, when a molecule, such as a variant or polymorphism of 238P1B2 is expressed in cancerous tissues, it is used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Example 45 Identification and Confirmation of Potential Signal Transduction Pathways

Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, GPCRs have been reported to activate MAK cascades as well as G proteins, and have been associated with the EGFR pathway in epithelial cells (Naor, Z., et al, Trends Endocrinol Metab. 2000, 11:91; Vacca F et al, Cancer Res. 2000, 60:5310; Della Rocca G J et al, J Biol Chem. 1999, 274:13978). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 238P1B2 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 238P1B2, including phospholipid pathways such as P13K, AKT, etc.; adhesion and migration pathways, including FAK, Rho, Rac-1, etc.; and mitogenic/survival cascades such as ERK, p38, etc. (Cell Growth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913).

Several GPCRs have been shown to transactivate receptor tyrosine kinases associated with the cell membrane, such as the EGF receptor (EGFR) (Pierce K L et al, J Biol Chem. 2001, 276:23155; Nath D et al, J Cell Sci. 2001, 114:1213). In order to determine whether 238P1B2 signaling results in the activation of EGFR, cells are grown in media alone or in the presence of the EGFR inhibitor AG1517. EGFR phosphorylation is compared in control and treated cells. Similarly, cross talk between 238P1B2 and EGFR pathways is investigated.

To confirm that 238P1B2 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

-   -   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress     -   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation     -   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress     -   4. ARE-luc, androgen receptor; steroids/MAPK;         growth/differentiation/apoptosis     -   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis     -   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.

Signaling pathways activated by 238P1B2 are mapped and used for the identification and validation of therapeutic targets. When 238P1B2 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46 238P1B2 Functions as a GPCR

Sequence and homology analysis of 238P1B2 indicate that the 238P1B2 protein is a member of the olfactory receptor family of GPCR. Olfactory receptors are known to regulate biological responses by activating adenylate cyclase. In order to confirm that 238P1B2 functions as a GPCR and mediates the activation of adenylate cyclase, cAMP accumulation in PC3 and PC3-238P1B2 cells are compared in cells grown in the presence or absence of fetal bovine serum (FBS). The cells are lysed and intracellular concentration of cAMP are measured using a commercially available enzyme immunoassay (EIA). Calculations of cAMP concentrations were based on OD450 of the standard curve. Similarly, the same assay can be used to determine whether the induction of cAMP accumulation by 238P1B2 is inhibited by GPCR inhibitors such as pertussis toxin.

GPCR transmit their signal by activating trimeric G proteins. Once GPCRs are activated, the associated Ga subunit binds GTP, dissociates from the receptor and participates in downstream signaling events (Schild D and Restrepo D. Physiol Rev. 1998, 78:429-66). In order to determine that inhibition of Ga subunits has an effect on 238P1B2 mediated cell growth, the effect of Ga inhibitors on the proliferation of 3T3-238P1B2 and PC3-238P1B2 cells is investigated. Control and 238P1B2-expressing cells are grown in the presence or absence of suramin or its derivative NF 449 (Sigma). Cells are analyzed for proliferation using an MTT-like assay.

When 238P1B2 functions as a GPCR, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

The 238P1B2 gene can contribute to the growth of cancer cells. The role of 238P1B2 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate as well as NIH 3T3 cells engineered to stably express 238P1B2. Parental cells lacking 238P1B2 and cells expressing 238P1B2 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288). To confirm that 238P1B2 mediates enhanced proliferation by way of its GPCR activity, control cells and cells expressing 238P1B2 are grown in the presence or absence of pertussis toxin, and evaluated for their proliferative capability using the same assay described above.

To confirm the role of 238P1B2 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 238P1B2 are compared to NIH-3T3 cells expressing 238P1B2, using a soft agar assay under stringent and more permissive conditions (Song Z. et al., Cancer Res. (2000) 60:6730).

To confirm the role of 238P1B2 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including prostate and fibroblast cell lines lacking 238P1B2 are compared to cells expressing 238P1B2. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.

238P1B2 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 238P1B2 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 238P1B2, including normal and tumor prostate cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 238P1B2 can play a critical role in regulating tumor progression and tumor load.

When 238P1B2 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of phsophodiesterase inhibitors on endothelial cells, 238P1B2 plays a role in angiogenesis (DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays of endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 238P1B2 in angiogenesis, enhancement or inhibition, is confirmed.

For example, endothelial cells engineered to express 238P1B2 are evaluated using tube formation and proliferation assays. The effect of 238P1B2 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 238P1B2 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 238P1B2 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes

Example 49 Regulation of Transcription

The cell surface localization of 238P1B2 and its similarity to GPCRs indicate that 238P1B2 is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 238P1B2. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and 238P1B2-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.

Thus, 238P1B2 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 50 Involvement in Cell Adhesion

Cell adhesion plays a critical role in tissue colonization and metastasis. Based on the presence of a fibronectin repeat in its C-terminus, 238P1B2 can participate in cellular organization, and as a consequence affects cell adhesion and motility. To confirm that 238P1B2 regulates cell adhesion, control cells lacking 238P1B2 are compared to cells expressing 238P1B2, using techniques previously described (see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. In another embodiment, cells lacking or expressing 238P1B2 are analyzed for their ability to mediate cell-cell adhesion using similar experimental techniques as described above. Both of these experimental systems are used to identify proteins, antibodies and/or small molecules that modulate cell adhesion to extracellular matrix and cell-cell interaction. Cell adhesion plays a critical role in tumor growth, progression, and colonization, and 238P1B2 is involved in these processes. Thus, it serves as a diagnostic, prognostic, preventative and/or therapeutic modality.

Example 51 Protein-Protein Association

Several GPCRs have been shown to interact with other proteins, thereby regulating signal transduction, gene transcription, transformation and cell adhesion (Sexton P M et al, Cell Signal. 2001, 13:73; Turner C E, J Cell Sci. 2000, 23:4139). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 238P1B2. Immunoprecipitates from cells expressing 238P1B2 and cells lacking 238P1B2 are compared for specific protein-protein associations.

Studies are performed to confirm the extent of association of 238P1B2 with effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates etc. Studies comparing 238P1B2 positive and 238P1B2 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 238P1B2-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 238P1B2, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 238P1B2.

Thus it is found that 238P1B2 associates with proteins and small molecules. Accordingly, 238P1B2 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.

Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Tables:

TABLE I Tissues that Express 238P1B2 When Malignant Prostate

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV (A) POSITION POSITION POSITION C Terminus 2 (Primary Anchor) 3 (Primary Anchor) (Primary Anchor) SUPER- MOTIFS A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWYMIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIW A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMFW YAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY D preferred motif b LIVMFAY DNQEST KRH preferred DR MFLIVWY VMSTACPLI Supermotif Italicized residues indicate less preferred or “tolerated” residues

TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS POSITION: 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° Anchor LIVMATQ LIVMAT A3 preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5) (4/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 preferred FWY (5/5) 1° Anchor FWY FWT 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA deleterious DE (3/5); DE G QN (DE) P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLA

TABLE IV (E) HLA Class I Motifs POSITION: C- 1 2 3 4 5 6 7 8 9 terminus or C- terminus A1 preferred GFYW 1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer STM Y deleterious DE RHKL A G A IVMP A1 preferred GRHK ASTC 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer LIVM DEAS Y deleterious A RHKDEPY DE PQN RHK PG GP FW A1 preferred YFW 1° Anchor DEAQN A YFWQN PASTC GDE P 1° Anchor 10-mer STM Y deleterious GP RHKG DE RHK QNA RHKY RHK A LIVM FW A1 preferred YFW STCL 1° Anchor A YFW PG G YFW 1° Anchor 10-mer IVM DEAS Y deleterious RHK RHKD P G PRHK QN EPY FW A2.1 preferred YFW 1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT VLIMAT deleterious DEP DERKH RKH DERKH A2.1 preferred AYFW 1° Anchor LVIM G G FYWL 1° Anchor 10-mer LMIVQAT VIM LMIVQAT deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK 1° Anchor YFW PRHK A YFW P 1° Anchor LMVISA YFW KYRHFA TFCGD deleterious DEP DE A11 preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor VTLMIS KRYH AGNCDF deleterious DEP A G A24 preferred YFWRHK 1° Anchor STC YFW YFW 1° Anchor 9-mer YFWM FLIW deleterious DEG DE G QNP DERHK G AQN A24 preferred 1° Anchor P YFWP P 1° Anchor 10-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 preferred RHK 1° Anchor YFW P YFW YFW AP 1° Anchor MVTALIS RK deleterious DEP DE ADE DE DE DE A3301 preferred 1° Anchor YFW AYFW 1° Anchor MVALFI RK ST deleterious GP DE A6801 preferred YFWSTC 1° Anchor YFWL YFW P 1° Anchor AVTMSLI IVM RK deleterious GP DEG RHK A B0702 preferred RHKFW 1° Anchor RHK RHK RHK RHK PA 1° Anchor Y P LMFWYAIV deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV 1° Anchor FWY FWY 1° Anchor M P LMGWYIV A deleterious AGP G G B51 preferred LIVMFW 1° Anchor FWY STC FWY G FWY 1° Anchor Y P LIVF WYAM deleterious AGPDER DE G DEQN GDE HKSTC B5301 preferred LIVMFW 1° Anchor FWY STC FWY LIVM FWY 1° Anchor Y P FWY IMFWYAL V deleterious AGPQN G RHKQN DE B5401 preferred FWY 1° Anchor FWYL LIVM ALIVM FWYAP 1° Anchor P IVM ATIVLMF WY deleterious GPQNDE GDES RHKDE DE QNDGE DE TC Italicized residues indicate less preferred or “tolerated” residues. The information in this Table is specific for 9-mers unless otherwise specified.

TABLE V V1-A1-9mers: 238P1B2 SEQ Pos 123456789 Score ID 152 DLLLILLSY 2.500 19. 66 VAVSNPLRY 2.500 20. 150 GVDLLLILL 2.500 21. 78 LTDSRIAQI 2.500 22. 102 VALLIRLSY 2.500 23. 186 HIVAFAIYY 2.500 24. 172 SPEERKETF 2.250 25. 51 VMESSVLLA 2.250 26. 200 LSIVHRFGK 1.500 27. 10 ATDLGLSIS 1.250 28. 21 VTMLSIFWF 1.250 29. 131 CTDTRINSA 1.250 30. 39 CLSHMFFIK 1.000 31. 114 QVLHHSYCY 1.000 32. 40 LSHMFFIKF 0.750 33. 112 HSQVLHHSY 0.750 34. 54 SSVLLAMAF 0.750 35. 123 HPDVMKLSC 0.625 36. 37 NACLSHMFF 0.500 37. 56 VLLAMAFDR 0.500 38. 157 LLSYVLIIR 0.500 39. 120 YCYHPDVMK 0.400 40. 68 VSNPLRYAM 0.300 41. 226 SPLMNPVIY 0.250 42. 65 FVAVSNPLR 0.200 43. 85 QIGVASVIR 0.200 44. 197 LISLSIVHR 0.200 45. 97 MLTPMVALL 0.200 46. 138 SAVGLTAMF 0.200 47. 182 TCVSHIVAF 0.200 48. 169 SVASPEERK 0.200 49. 168 LSVASPEER 0.150 50. 198 ISLSIVHRF 0.150 51. 24 LSIFWFNVR 0.150 52. 18 STLVTMLSI 0.125 53. 99 TPMVALLIR 0.125 54. 98 LTPMVALLI 0.125 55. 181 STCVSHIVA 0.125 56. 147 STVGVDLLL 0.125 57. 224 LISPLMNPV 0.100 58. 19 TLVTMLSIF 0.100 59. 57 LLAMAFDRF 0.100 60. 74 YAMILTDSR 0.100 61. 228 LMNPVIYSV 0.100 62. 158 LSYVLIIRT 0.075 63. 89 ASVIRGLLM 0.075 64. 184 VSHIVAFAI 0.075 65. 17 ISTLVTMLS 0.075 66. 34 ISFNACLSH 0.075 67. 77 ILTDSRIAQ 0.050 68. 9 SATDLGLSI 0.050 69. 217 MIANTYLLI 0.050 70. 110 YCHSQVLHH 0.050 71. 185 SHIVAFAIY 0.050 72. 191 AIYYIPLIS 0.050 73. 235 SVKTKQIRR 0.050 74. 222 YLLISPLMN 0.050 75. 130 SCTDTRINS 0.050 76. 20 LVTMLSIFW 0.050 77. 14 GLSISTLVT 0.050 78. 38 ACLSHMFFI 0.050 79. 23 MLSIFWFNV 0.050 80. 194 YIPLISLSI 0.050 81. 7 MLSATDLGL 0.050 82. 95 LLMLTPMVA 0.050 83. 148 TVGVDLLLI 0.050 84. 229 MNPVIYSVK 0.050 85. 156 ILLSYVLII 0.050 86. 139 AVGLTAMFS 0.050 87. 218 IANTYLLIS 0.050 88. 243 RAVIKILHS 0.050 89. 178 ETFSTCVSH 0.050 90. 176 RKETFSTCV 0.045 91. 31 VREISFNAC 0.045 92. 15 LSISTLVTM 0.030 93. 208 KQAPAYVHT 0.030 94. 53 ESSVLLAMA 0.030 95. 225 ISPLMNPVI 0.030 96. 118 HSYCYHPDV 0.030 97. 215 HTMIANTYL 0.025 98. 142 LTAMFSTVG 0.025 99. 49 FTVMESSVL 0.025 100. 220 NTYLLISPL 0.025 101. 42 HMFFIKFFT 0.025 102. 28 WFNVREISF 0.025 103. 59 AMAFDRFVA 0.025 104. 214 VHTMIANTY 0.025 105. 36 FNACLSHMF 0.025 106. 121 CYHPDVMKL 0.025 107. 213 YVHTMIANT 0.020 108. 143 TAMFSTVGV 0.020 109. 50 TVMESSVLL 0.020 110. 83 IAQIGVASV 0.020 111. 161 VLIIRTVLS 0.020 112. 231 PVIYSVKTK 0.020 113. 60 MAFDRFVAV 0.020 114. 183 CVSHIVAFA 0.020 115. 154 LLILLSYVL 0.020 116. 187 IVAFAIYYI 0.020 117. 244 AVIKILHSK 0.020 118.

TABLE V V1B-A1-9mers: 238P1B2 SEQ Pos 123456789 Score ID 23 GLEAFHTWI 0.900 119. 59 LHEPMYYFL 0.900 120. 31 ISIPFCFLS 0.750 121. 8 NITSTSIIF 0.500 122. 43 LLGNSLILF 0.500 123. 57 PSLHEPMYY 0.375 124. 56 QPSLHEPMY 0.250 125. 19 TGVPGLEAF 0.250 126. 58 SLHEPMYYF 0.200 127. 9 ITSTSIIFL 0.125 128. 18 LTGVPGLEA 0.125 129. 11 STSIIFLLT 0.125 130. 3 TSTLQNITS 0.075 131. 10 TSTSIIFLL 0.075 132. 38 LSVTALLGN 0.075 133. 12 TSIIFLLTG 0.075 134. 37 FLSVTALLG 0.050 135. 32 SIPFCFLSV 0.050 136. 25 EAFHTWISI 0.050 137. 48 LILFATITQ 0.050 138. 42 ALLGNSLIL 0.050 139. 46 NSLILFATI 0.030 140. 27 FHTWISIPF 0.025 141. 4 STLQNITST 0.025 142. 54 ITQPSLHEP 0.025 143. 40 VTALLGNSL 0.025 144. 2 ITSTLQNIT 0.025 145. 52 ATITQPSLH 0.025 146. 29 TWISIPFCF 0.025 147. 20 GVPGLEAFH 0.020 148. 51 FATITQPSL 0.020 149. 16 FLLTGVPGL 0.020 150. 61 EPMYYFLSM 0.013 151. 21 VPGLEAFHT 0.013 152. 45 GNSLILFAT 0.013 153. 44 LGNSLILFA 0.013 154. 13 SIIFLLTGV 0.010 155. 1 FITSTLQNI 0.010 156. 35 FCFLSVTAL 0.010 157. 47 SLILFATIT 0.010 158. 41 TALLGNSLI 0.010 159. 39 SVTALLGNS 0.010 160. 30 WISIPFCFL 0.010 161. 5 TLQNITSTS 0.010 162. 53 TTTQPSLHE 0.005 163. 28 HTWISIPFC 0.005 164. 60 HEPMYYFLS 0.003 165. 33 IPFCFLSVT 0.003 166. 7 QNITSTSII 0.003 167. 55 TQPSLHEPM 0.002 168. 6 LQNITSTSI 0.002 169. 14 IIFLLTGVP 0.001 170. 36 CFLSVTALL 0.001 171. 49 ILFATITQP 0.001 172. 15 IFLLTGVPG 0.001 173. 17 LLTGVPGLE 0.001 174. 50 LFATITQPS 0.001 175. 62 PMYYFLSML 0.001 176. 24 LEAFHTWIS 0.001 177. 22 PGLEAFHTW 0.000 178. 34 PFCFLSVTA 0.000 179. 26 AFHTWISTI 0.000 180. 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE V V2-A1-9mers: 238P1B2 SEQ Pos 123456789 Score ID 8 LTSPLMNPV 0.250 181. 1 MIANTYLLT 0.050 182. 2 IANTYLLTS 0.050 183. 6 YLLTSPLMN 0.050 184. 9 TSPLMNPVI 0.030 185. 4 NTYLLTSPL 0.025 186. 7 LLTSPLMNP 0.005 187. 5 TYLLTSPLM 0.001 188. 3 ANTYLLTSP 0.000 189.

TABLE VI V1-A1-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 10 ATDLGLSIST 6.250 190. 150 GVDLLLILLS 2.500 191. 65 FVAVSNPLRY 2.500 192. 101 MVALLIRLSY 2.500 193. 51 VMESSVLLAM 2.250 194. 225 ISPLMNPVIY 1.500 195. 184 VSHIVAFAIY 1.500 196. 78 LTDSRIAQIG 1.250 197. 98 LTPMVALLIR 1.250 198. 131 CTDTRTNSAV 1.250 199. 38 ACLSHMFFIK 1.000 200. 228 LMNPVIYSVK 1.000 201. 199 SLSIVHRFGK 1.000 202. 53 ESSVLLAMAF 0.750 203. 39 CLSHMFFIKF 0.500 204. 55 SVLLAMAFDR 0.500 205. 156 ILLSYVLIIR 0.500 206. 20 LVTMLSIFWF 0.500 207. 213 YVHTMIANTY 0.500 208. 97 MLTPMVALLI 0.500 209. 181 STCVSHIVAF 0.500 210. 68 VSNPLRYAMI 0.300 211. 168 LSVASPEERK 0.300 212. 137 NSAVGLTAMF 0.300 213. 18 STLVTMLSIF 0.250 214. 172 SPEERKETFS 0.225 215. 243 RAVIKILHSK 0.200 216. 224 LISPLMNPVI 0.200 217. 40 LSHMFFIKFF 0.150 218. 171 ASPEERKETF 0.150 219. 113 SQVLHHSYCY 0.150 220. 185 SHIVAFAIYY 0.125 221. 151 VDLLLILLSY 0.125 222. 36 FNACLSHMFF 0.125 223. 123 HPDVMKLSCT 0.125 224. 147 STVGVDLLLI 0.125 225. 170 VASPEERKET 0.100 226. 56 VLLAMAFDRF 0.100 227. 167 VLSVASPEER 0.100 228. 23 MLSIFWFNVR 0.100 229. 197 LISLSIVHRF 0.100 230. 31 VREISFNACL 0.090 231. 234 YSVKTKQIRR 0.075 232. 8 LSATDLGLSI 0.075 233. 180 FSTCVSHIVA 0.075 234. 146 FSTVGVDLLL 0.075 235. 17 ISTLVTMLSI 0.075 236. 129 LSCTDTRINS 0.075 237. 89 ASVIRGLLML 0.075 238. 204 HRFGKQAPAY 0.050 239. 94 GLLMLTPMVA 0.050 240. 85 QIGVASVIRG 0.050 241. 88 VASVIRGLLM 0.050 242. 220 NTYLLISPLM 0.050 243. 183 CVSHIVAFAI 0.050 244. 190 FAIYYIPLIS 0.050 245. 135 RINSAVGLTA 0.050 246. 76 MILTDSRIAQ 0.050 247. 37 NACLSHMFFI 0.050 248. 215 HTMIANTYLL 0.050 249. 217 MIANTYLLIS 0.050 250. 155 LILLSYVLII 0.050 251. 188 VAFAIYYIPL 0.050 252. 49 FTVMESSVLL 0.050 253. 148 TVGVDLLLIL 0.050 254. 50 TVMESSVLLA 0.050 255. 191 AIYYIPLISL 0.050 256. 16 SISTLVTMLS 0.050 257. 33 EISFNACLSH 0.050 258. 19 TLVTMLSIFW 0.050 259. 142 LTAMFSTVGV 0.050 260. 120 YCYHPDVMKL 0.050 261. 138 SAVGLTAMFS 0.050 262. 139 AVGLTAMFST 0.050 263. 230 NPVIYSVKTK 0.050 264. 161 VLIIRTVLSV 0.050 265. 58 LAMAFDRFVA 0.050 266. 157 LLSYVLIIRT 0.050 267. 176 RKETFSTCVS 0.045 268. 84 AQIGVASVIR 0.030 269. 108 LSYCHSQVLH 0.030 270. 178 ETFSTCVSHI 0.025 271. 6 SMLSATDLGL 0.025 272. 111 CHSQVLHHSY 0.025 273. 21 VTMLSIFWFN 0.025 274. 27 FWFNVREISF 0.025 275. 193 YYIPLISLSI 0.025 276. 42 HMFFIKFFTV 0.025 277. 216 TMIANTYLLI 0.025 278. 22 TMLSIFWFNV 0.025 279. 14 GLSISTLVTM 0.020 280. 119 SYCYHPDVMK 0.020 281. 103 ALLIRLSYCH 0.020 282. 182 TCVSHIVAFA 0.020 283. 60 MAFDRFVAVS 0.020 284. 153 LLLILLSYVL 0.020 285. 154 LLILLSYVLI 0.020 286. 160 YVLIIRTVLS 0.020 287. 82 RIAQIGVASV 0.020 288. 67 AVSNPLRYAM 0.020 289.

TABLE VI V1B-A1-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 59 LHEPMYYFLS 2.250 290. 23 GLEAFHTWIS 0.900 291. 31 ISIPFCFLSV 0.750 292. 56 QPSLHEPMYY 0.625 293. 42 ALLGNSLILF 0.500 294. 54 ITQPSLHEPM 0.250 295. 18 LTGVPGLEAF 0.250 296. 55 TQPSLHEPMY 0.150 297. 2 ITSTLQNITS 0.125 298. 7 QNITSTSIIF 0.125 299. 28 HTWISIPFCF 0.125 300. 9 ITSTSIIFLL 0.125 301. 11 STSIIFLLTG 0.125 302. 10 TSTSIIFLLT 0.075 303. 19 TGVPGLEAFH 0.050 304. 47 SLILFATITQ 0.050 305. 37 FLSVTALLGN 0.050 306. 20 GVPGLEAFHT 0.050 307. 30 WISIPFCFLS 0.050 308. 8 NITSTSIIFL 0.050 309. 43 LLGNSLILFA 0.050 310. 41 TALLGNSLIL 0.050 311. 17 LLTGVPGLEA 0.050 312. 57 PSLHEPMYYF 0.030 313. 40 VTALLGNSLI 0.025 314. 4 STLQNITSTS 0.025 315. 26 AFHTWISIPF 0.025 316. 58 SLHEPMYYFL 0.020 317. 35 FCFLSVTALL 0.020 318. 14 IIFLLTGVPG 0.020 319. 46 NSLILFATIT 0.015 320. 3 TSTLQNITST 0.015 321. 38 LSVTALLGNS 0.015 322. 12 TSIIFLLTGV 0.015 323. 52 ATITQPSLHE 0.013 324. 44 LGNSLILFAT 0.013 325. 1 FITSTLQNIT 0.010 326. 5 TLQNITSTSI 0.010 327. 32 SIPFCFLSVT 0.010 328. 39 SVTALLGNSL 0.010 329. 49 ILFATITQPS 0.010 330. 51 FATITQPSLH 0.010 331. 45 GNSLILFATI 0.005 332. 33 IPFCFLSVTA 0.005 333. 60 HEPMYYFLSM 0.003 334. 24 LEAFHTWISI 0.003 335. 36 CFLSVTALLG 0.003 336. 21 VPGLEAFHTW 0.003 337. 61 EPMYYFLSML 0.003 338. 62 PMYYFLSMLS 0.003 339. 6 LQNITSTSII 0.002 340. 27 FHTWISIPFC 0.001 341. 16 FLLTGVPGLE 0.001 342. 13 SIIFLLTGVP 0.001 343. 50 LFATITQPSL 0.001 344. 25 EAFHTWISIP 0.001 345. 48 LILFATITQP 0.001 346. 15 IFLLTGVPGL 0.001 347. 53 TITQPSLHEP 0.001 348. 29 TWISIPFCFL 0.001 349. 22 PGLEAFHTWI 0.000 350. 34 PFCFLSVTAL 0.000 351. 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE VI V2-A1-10mers SEQ Pos 1234567890 Score ID 10 TSPLMNPVIY 1.500 352. 9 LTSPLMNPVI 0.500 353. 5 NTYLLTSPLM 0.050 354. 2 MIANTYLLTS 0.050 355. 1 TMIANTYLLT 0.025 356. 8 LLTSPLMNPV 0.010 357. 7 YLLTSPLMNP 0.005 358. 6 TYLLTSPLMN 0.003 359. 4 ANTYLLTSPL 0.003 360. 3 IANTYLLTSP 0.001 361.

TABLE VII V1-A2-9mers: 238P1B2 SEQ Pos 123456789 Score ID 153 LLLILLSYV 5534.148 362. 23 MLSIFWFNV 4430.156 363. 94 GLLMLTPMV 257.342 364. 103 ALLIRLSYC 232.527 365. 96 LMLTPMVAL 223.203 366. 228 LMNPVIYSV 196.407 367. 141 GLTAMFSTV 132.149 368. 97 MLTPMVALL 83.527 369. 107 RLSYCHSQV 69.552 370. 156 ILLSYVLII 61.882 371. 60 MAFDRFVAV 47.333 372. 224 LISPLMNPV 37.393 373. 162 LIIRTVLSV 37.393 374. 128 KLSCTDTRI 36.515 375. 7 MLSATDLGL 36.316 376. 50 TVMESSVLL 28.356 377. 58 LAMAFDRFV 25.398 378. 42 HMFFIKFFT 21.598 379. 187 IVAFAIYYI 19.631 380. 38 ACLSHMFFI 18.488 381. 22 TMLSIFWFN 15.607 382. 216 TMIANTYLL 15.428 383. 154 LLILLSYVL 14.890 384. 155 LILLSYVLI 13.535 385. 160 YVLIIRTVL 13.044 386. 95 LLMLTPMVA 12.812 387. 59 AMAFDRFVA 8.532 388. 75 AMILTDSRI 7.535 389. 213 YVHTMIANT 6.899 390. 16 SISTLVTML 6.756 391. 194 YIPLISLSI 6.599 392. 183 CVSHIVAFA 5.499 393. 90 SVIRGLLML 4.299 394. 140 VGLTAMFST 4.082 395. 208 KQAPAYVHT 3.967 396. 83 IAQIGVASV 3.777 397. 12 DLGLSISTL 3.685 398. 217 MIANTYLLI 3.658 399. 143 TAMFSTVGV 3.574 400. 43 MFFIKFFTV 3.348 401. 14 GLSISTLVT 3.055 402. 13 LGLSISTLV 2.856 403. 202 IVHRFGKQA 2.808 404. 113 SQVLHHSYC 2.770 405. 195 IPLISLSIV 2.693 406. 220 NTYLLISPL 2.184 407. 148 TVGVDLLLI 2.100 408. 150 GVDLLLILL 1.720 409. 32 REISFNACL 1.578 410. 190 FAIYYIPLI 1.533 411. 222 YLLISPLMN 1.268 8912 18 STLVTMLSI 1.233 8913 67 AVSNPLRYA 1.100 412. 115 VLHHSYCYH 1.045 413. 149 VGVDLLLIL 0.917 414. 51 VMESSVLLA 0.898 415. 5 LSMLSATDL 0.877 416. 87 GVASVIRGL 0.860 417. 180 FSTCVSHIV 0.856 418. 30 NVREISFNA 0.786 419. 100 PMVALLIRL 0.781 420. 146 FSTVGVDLL 0.723 421. 184 VSHIVAFAI 0.671 422. 158 LSYVLIIRT 0.609 423. 9 SATDLGLSI 0.594 424. 49 FTVMESSVL 0.560 425. 56 VLLAMAFDR 0.544 426. 21 VTMLSIFWF 0.478 427. 84 AQIGVASVI 0.434 428. 206 FGKQAPAYV 0.402 429. 52 MESSVLLAM 0.378 430. 76 MILTDSRIA 0.352 431. 126 VMKLSCTDT 0.320 432. 108 LSYCHSQVL 0.311 433. 135 RINSAVGLT 0.306 434. 91 VIRGLLMLT 0.304 435. 147 STVGVDLLL 0.297 436. 93 RGLLMLTPM 0.276 437. 240 QIRRAVIKI 0.251 438. 98 LTPMVALLI 0.246 439. 78 LTDSRIAQI 0.227 440. 48 FFTVMESSV 0.222 441. 70 NPLRYAMIL 0.212 442. 215 HTMIANTYL 0.205 443. 3 YFLSMLSAT 0.203 444. 171 ASPEERKET 0.199 445. 209 QAPAYVHTM 0.159 446. 15 LSISTLVTM 0.127 447. 104 LLIRLSYCH 0.127 448. 161 VLIIRTVLS 0.127 449. 57 LLAMAFDRF 0.118 450. 77 ILTDSRIAQ 0.104 451. 69 SNPLRYAMI 0.102 452. 6 SMLSATDLG 0.098 453. 132 TDTRINSAV 0.097 454. 163 IIRTVLSVA 0.083 455. 199 SLSIVHRFG 0.082 456. 227 PLMNPVIYS 0.077 457. 137 NSAVGLTAM 0.075 458. 19 TLVTMLSIF 0.070 459.

TABLE VII V1B-A2-9mers: 238P1B2 SEQ Pos 123456789 Score ID 16 FLLTGVPGL 836.253 460. 30 WISIPFCFL 141.197 461. 42 ALLGNSLIL 32.407 462. 13 SIIFLLTGV 21.996 463. 32 SIPFCFLSV 18.170 464. 1 FITSTLQNI 15.177 465. 23 GLEAFHTWI 13.955 466. 47 SLILFATIT 11.610 467. 62 PMYYFLSML 9.493 468. 9 ITSTSIIFL 6.381 469. 35 FCFLSVTAL 5.459 470. 58 SLHEPMYYF 3.865 471. 10 TSTSIIFLL 1.860 472. 6 LQNITSTSI 1.798 473. 51 FATITQPSL 1.365 474. 33 IPFCFLSVT 1.096 475. 4 STLQNITST 0.881 476. 44 LGNSLILFA 0.697 477. 45 GNSLILFAT 0.649 478. 41 TALLGNSLI 0.536 479. 40 VTALLGNSL 0.504 480. 21 VPGLEAFHT 0.480 481. 46 NSLILFATI 0.479 482. 37 FLSVTALLG 0.343 483. 43 LLGNSLILF 0.291 484. 55 TQPSLHEPM 0.247 485. 28 HTWISIPFC 0.246 486. 49 TLFATITQP 0.216 487. 11 STSIIFLLT 0.197 488. 18 LTGVPGLEA 0.117 489. 5 TLQNITSTS 0.075 490. 2 ITSTLQNIT 0.072 491. 25 EAFHTWISI 0.068 492. 36 CFLSVTALL 0.055 493. 20 GVPGLEAFH 0.036 494. 7 QNITSTSII 0.028 495. 31 ISIPFCFLS 0.018 496. 61 EPMYYFLSM 0.017 497. 59 LHEPMYYFL 0.013 498. 48 LILFATITQ 0.013 499. 24 LEAFHTWIS 0.008 500. 12 TSIIFLLTG 0.004 501. 8 NITSTSIIF 0.004 502. 17 LLTGVPGLE 0.003 503. 53 TITQPSLHE 0.002 504. 39 SVTALLGNS 0.002 505. 38 LSVTALLGN 0.002 506. 52 ATITQPSLH 0.001 507. 54 ITQPSLHEP 0.001 508. 14 IIFLLTGVP 0.001 509. 19 TGVPGLEAF 0.001 510. 3 TSTLQNITS 0.000 511. 56 QPSLHEPMY 0.000 512. 60 HEPMYYFLS 0.000 513. 15 IFLLTGVPG 0.000 514. 22 PGLEAFHTW 0.000 515. 27 FHTWISIPF 0.000 516. 57 PSLHEPMYY 0.000 517. 50 LFATITQPS 0.000 518. 34 PFCFLSVTA 0.000 519. 29 TWISIPFCF 0.000 520. 26 AFHTWISIP 0.000 521. 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE VII V2-A2-9mers: 238P1B2 SEQ Pos 123456789 Score ID 8 LTSPLMNPV 3.777 522. 1 MIANTYLLT 2.613 523. 6 YLLTSPLMN 1.268 524. 4 NTYLLTSPL 0.949 525. 7 LLTSPLMNP 0.058 526. 9 TSPLMNPVI 0.028 527. 2 IANTYLLTS 0.004 528. 5 TYLLTSPLM 0.003 529. 3 ANTYLLTSP 0.000 530.

TABLE VIII V1-A2-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 22 TMLSIFWFNV 6963.743 531. 57 LLAMAFDRFV 494.237 532. 95 LLMLTPMVAL 309.050 533. 223 LLISPLMNPV 271.948 534. 161 VLIIRTVLSV 271.948 535. 152 DLLLILLSYV 244.154 536. 96 LMLTPMVALL 223.203 537. 77 ILTDSRIAQI 167.248 538. 4 FLSMLSATDL 98.267 539. 59 AMAFDRFVAV 95.441 540. 42 HMFFIKFFTV 69.637 541. 144 AMFSTVGVDL 57.085 542. 6 SMLSATDLGL 57.085 543. 153 LLLILLSYVL 55.091 544. 194 YIPLISLSIV 41.484 545. 157 LLSYVLIIRT 29.137 546. 154 LLILLSYVLI 26.604 547. 82 RIAQIGVASV 21.996 548. 191 AIYYIPLISL 21.619 549. 97 MLTPMVALLI 17.736 550. 50 TVMESSVLLA 15.167 551. 227 PLMNPVIYSV 13.022 552. 14 GLSISTLVTM 11.426 553. 216 TMIANTYLLI 11.302 554. 37 NACLSHMFFI 10.631 555. 107 RLSYCHSQVL 8.759 556. 139 AVGLTAMFST 8.698 557. 239 KQIRRAVIKI 8.515 558. 155 LILLSYVLII 8.509 559. 232 VIYSVKTKQI 7.804 560. 94 GLLMLTPMVA 7.536 561. 102 VALLIRLSYC 5.490 562. 140 VGLTAMFSTV 5.426 563. 183 CVSHIVAFAI 5.295 564. 12 DLGLSISTLV 5.216 565. 120 YCYHPDVMKL 4.721 566. 208 KQAPAYVHTM 4.055 567. 188 VAFAIYYIPL 2.799 568. 201 SIVHRFGKQA 2.596 569. 74 YAMILTDSRI 2.466 570. 25 SIFWFNVREI 2.442 571. 149 VGVDLLLILL 2.236 572. 148 TVGVDLLLIL 1.763 573. 93 RGLLMLTPMV 1.680 574. 142 LTAMFSTVGV 1.642 575. 90 SVIRGLLMLT 1.500 576. 49 ETVMESSVLL 1.365 577. 99 TPMVALLIRL 1.187 578. 158 LSYVLIIRTV 1.136 579. 162 LIIRTVLSVA 1.095 580. 58 LAMAFDRFVA 1.032 581. 224 LISPLMNPVI 1.000 582. 51 VMESSVLLAM 0.898 583. 29 FNVREISFNA 0.865 584. 20 LVTMLSIFWF 0.813 585. 135 RINSAVGLTA 0.683 586. 146 FSTVGVDLLL 0.641 587. 47 KFFTVMESSV 0.625 588. 186 HIVAFAIYYI 0.617 589. 41 SHMFFIKFFT 0.611 590. 222 YLLISPLMNP 0.583 591. 15 LSISTLVTML 0.545 592. 39 CLSHMFFIKF 0.538 593. 21 VTMLSIFWFN 0.510 594. 34 ISFNACLSHM 0.469 595. 56 VLLAMAFDRF 0.436 596. 67 AVSNPLRYAM 0.435 597. 70 NPLRYAMILT 0.414 598. 182 TCVSHIVAFA 0.410 599. 19 TLVTMLSIFW 0.410 600. 131 CTDTRINSAV 0.386 601. 52 MESSVLLAMA 0.378 602. 147 STVGVDLLLI 0.333 603. 219 ANTYLLISPL 0.321 604. 89 ASVIRGLLML 0.321 605. 130 SCTDTRINSA 0.306 606. 66 VAVSNPLRYA 0.297 607. 75 AMILTDSRIA 0.294 608. 103 ALLIRLSYCH 0.276 609. 17 ISTLVTMLSI 0.266 610. 205 RFGKQAPAYV 0.266 611. 30 NVREISFNAC 0.257 612. 11 TDLGLSISTL 0.252 613. 87 GVASVIRGLL 0.243 614. 220 NTYLLISPLM 0.221 615. 240 QIRRAVIKIL 0.211 616. 114 QVLHHSYCYH 0.199 617. 104 LLIRLSYCHS 0.190 618. 125 DVMKLSCTDT 0.181 619. 170 VASPEERKET 0.176 620. 68 VSNPLRYAMI 0.174 621. 209 QAPAYVHTMI 0.145 622. 214 VHTMIANTYL 0.139 623. 136 INSAVGLTAM 0.127 624. 13 LGLSISTLVT 0.125 625. 88 VASVIRGLLM 0.117 626. 141 GLTAMFSTVG 0.116 627. 8 LSATDLGLSI 0.116 628. 160 YVLIIRTVLS 0.111 629. 86 IGVASVIRGL 0.109 630.

TABLE VIII V1B-A2-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 58 SLHEPMYYFL 722.583 631. 43 LLGNSLILFA 106.837 632. 8 NITSTSIIFL 37.157 633. 5 TLQNITSTSI 10.433 634. 17 LLTGVPGLEA 8.446 635. 9 ITSTSIIFLL 6.729 636. 20 GVPGLEAFHT 3.782 637. 32 SIPFCFLSVT 3.672 638. 35 FCELSVTALL 2.373 639. 1 FITSTLQNIT 1.932 640. 39 SVTALLGNSL 1.869 641. 31 ISIPFCFLSV 1.466 642. 44 LGNSLILFAT 1.103 643. 12 TSIIFLLTGV 1.044 644. 30 WISIPFCFLS 1.039 645. 37 FLSVTALLGN 0.788 646. 6 LQNITSTSII 0.737 647. 42 ALLGNSLILF 0.634 648. 24 LEAFHTWISI 0.586 649. 22 PGLEAFHTWI 0.475 650. 49 ILFATITQPS 0.469 651. 41 TALLGNSLIL 0.450 652. 61 EPMYYFLSML 0.338 653. 46 NSLILFATIT 0.280 654. 40 VTALLGNSLI 0.246 655. 15 IFLLTGVPGL 0.215 656. 33 IPFCFLSVTA 0.204 657. 27 FHTWISIPFC 0.201 658. 45 GNSLILFATI 0.129 659. 3 TSTLQNITST 0.112 660. 10 TSTSIIFLLT 0.092 661. 14 IIFLLTGVPG 0.064 662. 54 ITQPSLHEPM 0.057 663. 16 FLLTGVPGLE 0.033 664. 29 TWISIPFCFL 0.031 665. 48 LILFATITQP 0.030 666. 50 LFATITQPSL 0.025 667. 47 SLILFATTTQ 0.015 668. 23 GLEAFHTWIS 0.015 669. 11 STSIIFLLTG 0.009 670. 55 TQPSLHEPMY 0.008 671. 60 HEPMYYFLSM 0.006 672. 51 FATITQPSLH 0.005 673. 53 TITQPSLHEP 0.005 674. 4 STLQNITSTS 0.004 675. 62 PMYYFLSMLS 0.004 676. 19 TGVPGLEAFH 0.003 677. 56 QPSLHEPMYY 0.003 678. 18 LTGVPGLEAF 0.002 679. 2 ITSTLQNITS 0.002 680. 28 HTWISIPFCF 0.002 681. 21 VPGLEAFHTW 0.002 682. 34 PFCFLSVTAL 0.001 683. 57 PSLHEPMYYF 0.001 684. 38 LSVTALLGNS 0.000 685. 52 ATITQPSLHE 0.000 686. 7 QNITSTSIIF 0.000 687. 36 CFLSVTALLG 0.000 688. 13 SIIFLLTGVP 0.000 689. 25 EAFHTWISIP 0.000 690. 26 AFHTWISTPF 0.000 691. 59 LHEPMYYFLS 0.000 692. 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE VIII V2-A2-10mers SEQ Pos 1234567890 Score ID 8 LLTSPLMNPV 271.948 693. 1 TMIANTYLLT 8.073 694. 7 YLLTSPLMNP 0.583 695. 5 NTYLLTSPLM 0.221 696. 4 ANTYLLTSPL 0.139 697. 9 LTSPLMNPVI 0.101 698. 2 MIANTYLLTS 0.040 699. 3 IANTYLLTSP 0.001 700. 10 TSPLMNPVIY 0.000 701. 6 TYLLTSPLMN 0.000 702.

TABLE IX V1-A3-9-mers: 238P1B2 SEQ Pos 123456789 Score ID 39 CLSHMFFIK 180.000 703. 157 LLSYVLIIR 18.000 704. 56 VLLANAFDR 18.000 705. 156 ILLSYVLII 16.200 706. 152 DLLLILLSY 8.100 707. 244 AVIKILHSK 6.750 708. 57 LLAMAFDRF 6.000 709. 19 TLVTMLSIF 4.500 710. 186 HIVAFAIYY 3.600 711. 169 SVASPEERK 3.000 712. 216 TMIANTYLL 2.700 713. 97 MLTPMVALL 2.700 714. 239 KQIRRAVIK 2.700 715. 154 LLILLSYVL 2.700 716. 42 HMFFIKFFT 2.250 717. 228 LMNPVIYSV 2.025 718. 96 LMLTPMVAL 2.025 719. 141 GLTAMFSTV 1.800 720. 23 MLSIFWFNV 1.800 721. 128 KLSCTDTRI 1.800 722. 114 QVLHHSYCY 1.800 723. 120 YCYHPDVMK 1.500 724. 51 VMESSVLLA 1.200 725. 7 MLSATDLGL 1.200 726. 104 LLIRLSYCH 0.900 727. 94 GLLMLTPMV 0.900 728. 75 AMTLTDSRI 0.900 729. 235 SVKTKQIRR 0.800 730. 21 VTMLSIFWF 0.675 731. 95 LLMLTPMVA 0.600 732. 197 LISLSIVHR 0.600 733. 59 AMAFDRFVA 0.600 734. 14 GLSISTLVT 0.600 735. 150 GVDLLLILL 0.540 736. 103 ALLIRLSYC 0.450 737. 153 LLLILLSYV 0.450 738. 200 LSIVHRFGK 0.450 739. 231 PVIYSVKTK 0.450 740. 22 TMLSIFWFN 0.405 741. 85 QIGVASVIR 0.400 742. 65 FVAVSNPLR 0.400 743. 240 QIRRAVIKI 0.360 744. 12 DLGLSISTL 0.270 745. 100 PMVALLIRL 0.270 746. 24 LSIFWFNVR 0.270 747. 90 SVIRGLLML 0.270 748. 187 IVAFAIYYI 0.270 749. 220 NTYLLISPL 0.225 750. 147 STVGVDLLL 0.203 751. 107 RLSYCHSQV 0.200 752. 115 VLHHSYCYH 0.200 753. 194 YIPLISLSI 0.180 754. 16 SISTLVTML 0.180 755. 148 TVGVDLLLI 0.180 756. 162 LIIRTVLSV 0.180 757. 40 LSHMFFIKF 0.180 758. 99 TPMVALLIR 0.180 759. 102 VALLIRLSY 0.180 760. 50 TVMESSVLL 0.135 761. 30 NVREISFNA 0.135 762. 160 YVLIIRTVL 0.135 763. 223 LLISPLMNP 0.135 764. 18 STLVTMLSI 0.135 765. 66 VAVSNPLRY 0.120 766. 217 MIANTYLLI 0.120 767. 126 VMKLSCTDT 0.100 768. 183 CVSHIVAFA 0.090 769. 98 LTPMVALLI 0.090 770. 144 AMFSTVGVD 0.090 771. 155 LILLSYVLI 0.090 772. 198 ISLSIVHRF 0.068 773. 84 AQIGVASVI 0.061 774. 229 MNPVIYSVK 0.060 775. 191 AIYYIPLIS 0.060 776. 196 PLISLSIVH 0.060 777. 161 VLIIRTVLS 0.060 778. 74 YAMILTDSR 0.060 779. 222 YLLISPLMN 0.060 780. 226 SPLMNPVIY 0.060 781. 227 PLMNPVIYS 0.054 782. 70 NPLRYAMIL 0.054 783. 163 IIRTVLSVA 0.045 784. 49 FTVMESSVL 0.045 785. 215 HTMIANTYL 0.045 786. 182 TCVSHIVAF 0.045 787. 91 VIRGLLMLT 0.045 788. 138 SAVGLTAMF 0.045 789. 78 LTDSRIAQI 0.045 790. 224 LISPLMNPV 0.045 791. 87 GVASVIRGL 0.041 792. 190 FAIYYIPLI 0.041 793. 38 ACLSHMFFI 0.041 794. 37 NACLSHMFF 0.040 795. 20 LVTMLSIFW 0.040 796. 77 ILTDSRIAQ 0.040 797. 234 YSVKTKQIR 0.030 798. 25 SIFWFNVRE 0.030 799. 6 SMLSATDLG 0.030 800. 202 IVHRFGKQA 0.030 801. 168 LSVASPEER 0.030 802.

TABLE IX V1B-A3-9mers: 238P1B2 SEQ ID Pos 123456789 Score 803 58 SLHEPMYYF 20.250 804 43 LLGNSLILF 6.000 805 23 GLEAFHTWI 5.400 806 42 ALLGNSLIL 2.700 807 16 FLLTGVPGL 2.700 808 62 PMYYFLSML 1.350 809 47 SLILFATIT 0.450 810 8 NITSTSIIF 0.400 811 30 WISIPFCFL 0.270 812 49 ILFATITQP 0.150 813 32 SIPFCFLSV 0.120 814 9 ITSTSIIFL 0.090 815 20 GVPGLEAFH 0.090 816 1 FITSTLQNI 0.090 817 35 FCFLSVTAL 0.090 818 28 HTWISIPFC 0.075 819 5 TLQNITSTS 0.060 820 11 STSIIFLLT 0.045 821 13 SIIFLLTGV 0.045 822 37 FLSVTALLG 0.040 823 56 QPSLHEPMY 0.040 824 18 LTGVPGLEA 0.030 825 40 VTALLGNSL 0.030 826 33 IPFCFLSVT 0.022 827 10 TSTSIIFLL 0.020 828 6 LQNITSTSI 0.018 829 25 EAFHTWISI 0.018 830 61 EPMYYFLSM 0.016 831 52 ATITQPSLH 0.015 832 29 TWISIPFCF 0.013 833 17 LLTGVPGLE 0.013 834 46 NSLILFATI 0.013 835 4 STLQNITST 0.011 836 41 TALLGNSLI 0.009 837 19 TGVPGLEAF 0.007 838 27 FHTWISIPF 0.006 839 57 PSLHEPMYY 0.006 840 51 FATITQPSL 0.006 841 53 TITQPSLHE 0.006 842 48 LILFATITQ 0.006 843 55 TQPSLHEPM 0.006 844 2 ITSTLQNIT 0.005 845 45 GNSLILFAT 0.004 846 31 ISIPFCFLS 0.004 847 21 VPGLEAFHT 0.003 848 14 IIFLLTGVP 0.003 849 59 LHEPMYYFL 0.003 850 54 ITQPSLHEP 0.002 851 7 QNITSTSII 0.002 852 39 SVTALLGNS 0.002 853. 36 CFLSVTALL 0.001 854. 12 TSIIFLLTG 0.001 855. 60 HEPMYYFLS 0.001 856. 44 LGNSLILFA 0.001 857. 38 LSVTALLGN 0.000 858. 3 TSTLQNITS 0.000 859. 24 LEAFHTWIS 0.000 860. 22 PGLEAFHTW 0.000 861. 26 AFHTWISIP 0.000 862. 50 LFATITQPS 0.000 863. 15 IFLLTGVPG 0.000 864. 34 PFCFLSVTA 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE IX V2-A3-9mers-238P1B2 SEQ Pos 123456789 Score ID 4 NTYLLTSPL 0.150 865 7 LLTSPLMNP 0.090 866 6 YLLTSPLMN 0.060 867 8 LTSPLMNPV 0.022 868 1 MIANTYLLT 0.020 869 2 IANTYLLTS 0.004 870 9 TSPLMNPVI 0.003 871 5 TYLLTSPLM 0.000 872 3 ANTYLLTSP 0.000 873

TABLE X V1-A3-10-mers: 238P1B2 SEQ Pos 1234567890 Score ID 199 SLSIVHRFGK 60.000 874 228 LMNPVIYSVK 45.000 875 39 CLSHMFFIKF 36.000 876 23 MLSIFWFNVR 36.000 877 156 ILLSYVLIIR 27.000 878 42 HMFFIKFFTV 9.000 879 56 VLLAMAFDRF 9.000 880 144 AMFSTVGVDL 4.500 881 167 VLSVASPEER 4.000 882 126 VMKLSCTDTR 4.000 883 153 LLLILLSYVL 2.700 884 38 ACLSHMFFIK 2.700 885 22 TMLSIFWFNV 2.700 886 96 LMLTPMVALL 2.700 887 97 MLTPMVALLI 2.700 888 95 LLMLTPMVAL 2.025 889 6 SMLSATDLGL 1.800 890 94 GLLMLTPMVA 1.800 891 161 VLIIRTVLSV 1.800 892 216 TMIANTYLLI 1.800 893 55 SVLLAMAFDR 1.800 894 155 LILLSYVLII 1.620 895 191 AIYYIPLISL 1.350 896 101 MVALLIRLSY 1.200 897 14 GLSISTLVTM 0.900 898 51 VMESSVLLAM 0.900 899 197 LISLSIVHRF 0.900 900 196 PLISLSIVHR 0.900 901 20 LVTMLSIFWF 0.900 902 103 ALLIRLSYCH 0.900 903 77 ILTDSRIAQI 0.900 904 154 LLILLSYVLI 0.900 905 65 FVAVSNPLRY 0.800 906 243 RAVIKILHSK 0.675 907 98 LTPMVALLIR 0.600 908 4 FLSMLSATDL 0.600 909 107 RLSYCHSQVL 0.600 910 213 YVHTMIANTY 0.600 911 19 TLVTMLSIFW 0.600 912 59 AMAFDRFVAV 0.600 913 113 SQVLHHSYCY 0.540 914 239 KQIRRAVIKI 0.486 915 223 LLISPLMNPV 0.450 916 230 NPVIYSVKTK 0.450 917 186 HIVAFAIYYI 0.405 918 157 LLSYVLIIRT 0.300 919 84 AQIGVASVIR 0.270 920 50 TVMESSVLLA 0.270 921 183 CVSHIVAFAI 0.270 922 208 KQAPAYVHTM 0.243 923 18 STLVTMLSIF 0.225 924 168 LSVASPEERK 0.225 925 147 STVGVDLLLI 0.203 926 227 PLMNPVIYSV 0.203 927 148 TVGVDLLLIL 0.180 928 120 YCYHPDVMKL 0.180 929 115 VLHHSYCYHP 0.180 930 188 VAFAIYYIPL 0.180 931 141 GLTAMESTVG 0.180 932 181 STCVSHIVAF 0.150 933 232 VIYSVKTKQI 0.150 934 215 HTMIANTYLL 0.135 935 152 DLLLILLSYV 0.135 936 222 YLLISPLMNP 0.135 937 178 ETFSTCVSHI 0.135 938 30 NVREISFNAC 0.090 939 12 DLGLSISTLV 0.090 940 224 LISPLMNPVI 0.090 941 237 KTKQIRRAVI 0.090 942 25 SIFWFNVREI 0.090 943 87 GVASVIRGLL 0.081 944 220 NTYLLISPLM 0.075 945 240 QIRRAVIKIL 0.068 946 162 LIIRTVLSVA 0.068 947 135 RINSAVGLTA 0.060 948 67 AVSNPLRYAM 0.060 949 104 LLIRLSYCHS 0.060 950 57 LLAMAFDRFV 0.060 951 234 YSVKTKQIRR 0.060 952 184 VSHIVAFAIY 0.060 953 82 RIAQIGVASV 0.060 954 201 SIVHREGKQA 0.045 955 139 AVGLTAMFST 0.045 956 90 SVIRGLLMLT 0.045 957 49 FTVMESSVLL 0.045 958 185 SHIVAFAIYY 0.036 959 217 MIANTYLLIS 0.036 960 150 GVDLLLILLS 0.036 961 119 SYCYHPDVMK 0.030 962 194 YIPLISLSIV 0.030 963 114 QVLHHSYCYH 0.030 964 75 AMILTDSRIA 0.030 965 204 HRFGKQAPAY 0.030 966 37 NACLSHMFFI 0.027 967 99 TPMVALLIRL 0.027 968 151 VDLLLILLSY 0.027 969 45 FIKFFTVMES 0.024 970 40 LSHMFFIKFF 0.022 971 21 VTMLSIFWFN 0.020 972 238 TKQIRRAVIK 0.020 973

TABLE X V1B-A3-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 42 ALLGNSLILF 13.500 974 58 SLHEPMYYFL 2.700 975 28 HTWISIPFCF 2.250 976 17 LLTGVPGLEA 0.900 977 43 LLGNSLILFA 0.600 978 5 TLQNITSTSI 0.600 979 23 GLEAFHTWIS 0.360 980 49 ILFATITQPS 0.300 981 9 ITSTSIIFLL 0.203 982 8 NITSTSIIFL 0.180 983 18 LTGVPGLEAF 0.150 984 20 GVPGLEAFHT 0.135 985 55 TQPSLHEPMY 0.120 986 56 QPSLHEPMYY 0.080 987 39 SVTALLGNSL 0.060 988 47 SLILFATITQ 0.060 989 37 FLSVTALLGN 0.060 990 32 SIPFCFLSVT 0.045 991 35 FCFLSVTALL 0.045 992 62 PMYYFLSMLS 0.040 993 30 WISIPFCFLS 0.036 994 40 VTALLGNSLI 0.030 995 6 LQNITSTSII 0.018 996 41 TALLGNSLIL 0.018 997 54 ITQPSLHEPM 0.015 998 16 FLLTGVPGLE 0.013 999 31 ISIPFCFLSV 0.013 1000 61 EPMYYFLSML 0.012 1001 7 QNITSTSIIF 0.012 1002 45 GNSLILFATI 0.011 1003 60 HEPMYYFLSM 0.011 1004 57 PSLHEPMYYF 0.010 1005 1 FITSTLQNIT 0.010 1006 14 IIFLLTGVPG 0.010 1007 33 IPFCFLSVTA 0.010 1008 11 STSIIFLLTG 0.009 1009 26 AFHTWISIPF 0.006 1010 21 VPGLEAFHTW 0.006 1011 10 TSTSIIFLLT 0.005 1012 52 ATITQPSLHE 0.005 1013 4 STLQNITSTS 0.005 1014 48 LILFATITQP 0.005 1015 29 TWISIPFCFL 0.004 1016 2 ITSTLQNITS 0.004 1017 24 LEAFHTWISI 0.004 1018 53 TITQPSLHEP 0.003 1019 15 IFLLTGVPGL 0.003 1020 46 NSLILFATIT 0.002 1021 12 TSIIFLLTGV 0.002 1022 51 FATITQPSLH 0.002 1023 25 EAFHTWISIP 0.001 1024 13 SIIFLLTGVP 0.001 1025 3 TSTLQNITST 0.001 1026 44 LGNSLILFAT 0.001 1027 50 LFATITQPSL 0.001 1028 59 LHEPMYYFLS 0.001 1029 19 TGVPGLEAFH 0.000 1030 22 PGLEAFHTWI 0.000 1031 27 FHTWISIPFC 0.000 1032 34 PFCFLSVTAL 0.000 1033 38 LSVTALLGNS 0.000 1034 36 CFLSVTALLG 0.000 1035 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE X V2-A3-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 1 TMIANTYLLT 0.300 1036 8 LLTSPLMNPV 0.300 1037 7 YLLTSPLMNP 0.135 1038 5 NTYLLTSPLM 0.050 1039 9 LTSPLMNPVI 0.045 1040 2 MIANTYLLTS 0.036 1041 10 TSPLMNPVIY 0.020 1042 4 ANTYLLTSPL 0.001 1043 3 IANTYLLTSP 0.000 1044 6 TYLLTSPLMN 0.000 1045

TABLE XI V1-A11-9mers: 238P1B2 SEQ Pos 123456789 Score ID 244 AVIKILHSK 3.000 1046 239 KQIRRAVIK 2.700 1047 169 SVASPEERK 2.000 1048 39 CLSHMFFIK 1.200 1049 235 SVKTKQIRR 0.800 1050 65 FVAVSNPLR 0.400 1051 120 YCYHPDVMK 0.400 1052 56 VLLAMAFDR 0.360 1053 157 LLSYVLIIR 0.160 1054 99 TPMVALLIR 0.160 1055 231 PVIYSVKTK 0.150 1056 150 GVDLLLILL 0.120 1057 200 LSIVHRFGK 0.090 1058 197 LISLSIVHR 0.080 1059 74 YAMILTDSR 0.080 1060 85 QIGVASVIR 0.080 1061 30 NVREISFNA 0.060 1062 21 VTMLSIFWF 0.060 1063 114 QVLHHSYCY 0.060 1064 90 SVIRGLLML 0.060 1065 148 TVGVDLLLI 0.040 1066 20 LVTMLSIFW 0.040 1067 187 IVAFAIYYI 0.040 1068 50 TVMESSVLL 0.040 1069 229 MNPVIYSVK 0.040 1070 147 STVGVDLLL 0.030 1071 18 STLVTMLSI 0.030 1072 160 YVLIIRTVL 0.030 1073 215 HTMIANTYL 0.020 1074 181 STCVSHIVA 0.020 1075 220 NTYLLISPL 0.020 1076 98 LTPMVALLI 0.020 1077 183 CVSHIVAFA 0.020 1078 94 GLLMLTPMV 0.018 1079 95 LLMLTPMVA 0.016 1080 2 YYFLSMLSA 0.016 1081 192 IYYIPLISL 0.016 1082 49 FTVMESSVL 0.015 1083 43 MFFIKFFTV 0.012 1084 23 MLSIFWFNV 0.012 1085 59 AMAFDRFVA 0.012 1086 128 KLSCTDTRI 0.012 1087 141 GLTAMFSTV 0.012 1088 154 LLILLSYVL 0.012 1089 186 HIVAFAIYY 0.012 1090 104 LLIRLSYCH 0.012 1091 107 RLSYCHSQV 0.012 1092 162 LIIRTVLSV 0.012 1093 216 TMIANTYLL 0.012 1094 156 ILLSYVLII 0.012 1095 131 CTDTRINSA 0.010 1096 78 LTDSRIAQI 0.010 1097 202 IVHRFGKQA 0.010 1098 38 ACLSHMFFI 0.009 1099 64 RFVAVSNPL 0.009 1100 84 AQIGVASVI 0.009 1101 217 MIANTYLLI 0.008 1102 7 MLSATDLGL 0.008 1103 240 QIRRAVIKI 0.008 1104 194 YIPLISLSI 0.008 1105 228 LMNPVTYSV 0.008 1106 121 CYHPDVMKL 0.008 1107 51 VMESSVLLA 0.008 1108 168 LSVASPEER 0.006 1109 127 MKLSCTDTR 0.006 1110 234 YSVKTKQIR 0.006 1111 24 LSIFWFNVR 0.006 1112 87 GVASVIRGL 0.006 1113 19 TLVTMLSIF 0.006 1114 205 RFGKQAPAY 0.006 1115 221 TYLLISPLM 0.006 1116 70 NPLRYAMIL 0.006 1117 155 LILLSYVLI 0.006 1118 178 ETFSTCVSH 0.006 1119 75 AMILTDSRI 0.006 1120 66 VAVSNPLRY 0.006 1121 102 VALLIRLSY 0.006 1122 96 LMLTPMVAL 0.006 1123 153 LLLILLSYV 0.006 1124 165 RTVLSVASP 0.005 1125 57 LLAMAFDRF 0.004 1126 28 WFNVREISF 0.004 1127 163 IIRTVLSVA 0.004 1128 139 AVGLTAMFS 0.004 1129 143 TAMFSTVGV 0.004 1130 60 MAFDRFVAV 0.004 1131 16 SISTLVTML 0.004 1132 110 YCHSQVLHH 0.004 1133 37 NACLSHMFF 0.004 1134 109 SYCHSQVLH 0.004 1135 119 SYCYHPDVM 0.004 1136 189 AFAIYYIPL 0.004 1137 9 SATDLGLSI 0.004 1138 224 LISPLMNPV 0.004 1139 115 VLHHSYCYH 0.004 1140 97 MLTPMVALL 0.004 1141 152 DLLLILLSY 0.004 1142 237 KTKQIRRAV 0.003 1143 55 SVLLAMAFD 0.003 1144 138 SAVGLTAMF 0.003 1145

TABLE XI V1B-A11-9mers: 238P1B2 SEQ Pos 123456789 Score ID 20 GVPGLEAFH 0.060 1146 9 ITSTSIIFL 0.020 1147 18 LTGVPGLEA 0.020 1148 52 ATITQPSLH 0.015 1149 42 ALLGNSLIL 0.012 1150 23 GLEAFHTWI 0.012 1151 40 VTALLGNSL 0.010 1152 43 LLGNSLILF 0.008 1153 8 NITSTSIIF 0.008 1154 58 SLHEPMYYF 0.008 1155 32 SIPFCFLSV 0.008 1156 55 TQPSLHEPM 0.006 1157 6 LQNITSTSI 0.006 1158 16 FLLTGVPGL 0.006 1159 13 SIIFLLTGV 0.006 1160 1 FITSTLQNI 0.004 1161 35 FCFLSVTAL 0.004 1162 30 WISIPFCFL 0.004 1163 41 TALLGNSLI 0.003 1164 36 CFLSVTALL 0.003 1165 25 EAFHTWISI 0.002 1166 61 EPMYYFLSM 0.002 1167 51 FATITQPSL 0.002 1168 39 SVTALLGNS 0.002 1169 56 QPSLHEPMY 0.002 1170 28 HTWISIPFC 0.002 1171 11 STSIIFLLT 0.002 1172 4 STLQNITST 0.002 1173 48 LILFATITQ 0.001 1174 2 ITSTLQNIT 0.001 1175 54 ITQPSLHEP 0.001 1176 29 TWISIPFCF 0.001 1177 37 FLSVTALLG 0.001 1178 14 IIFLLTGVP 0.001 1179 62 PMYYFLSML 0.001 1180 49 ILFATITQP 0.001 1181 53 TITQPSLHE 0.001 1182 7 QNITSTSII 0.001 1183 47 SLILFATIT 0.001 1184 10 TSTSIIFLL 0.001 1185 21 VPGLEAFHT 0.001 1186 44 LGNSLILFA 0.000 1187 59 LHEPMYYFL 0.000 1188 5 TLQNITSTS 0.000 1189 27 FHTWISIPF 0.000 1190 33 IPFCFLSVT 0.000 1191 17 LLTGVPGLE 0.000 1192 45 GNSLILFAT 0.000 1193 19 TGVPGLEAF 0.000 1194 46 NSLILFATI 0.000 1195 15 IFLLTGVPG 0.000 1196 34 PFCFLSVTA 0.000 1197 26 AFHTWISIP 0.000 1198 50 LFATITQPS 0.000 1199 60 HEPMYYFLS 0.000 1200 24 LEAFHTWIS 0.000 1201 31 ISTPFCFLS 0.000 1202 38 LSVTALLGN 0.000 1203 57 PSLHEPMYY 0.000 1204 12 TSIIFLLTG 0.000 1205 3 TSTLQNITS 0.000 1206 22 PGLEAFHTW 0.000 1207 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE XI V2-A11-9mers SEQ Pos 123456789 Score ID 4 NTYLLTSPL 0.020 1208 8 LTSPLMNPV 0.010 1209 5 TYLLTSPLM 0.006 1210 6 YLLTSPLMN 0.001 1211 7 LLTSPLMNP 0:001 1212 1 MIANTYLLT 0.001 1213 2 IANTYLLTS 0.000 1214 9 TSPLMNPVI 0.000 1215 3 ANTYLLTSP 0.000 1216

TABLE XII V1-A11-10-mers: 238P1B2 SEQ Pos 1234567890 Score ID 55 SVLLAMAFDR 1.800 1217 199 SLSIVHRFGK 1.200 1218 38 ACLSHMFFIK 0.900 1219 243 RAVIKILHSK 0.900 1220 98 LTPMVALLIR 0.400 1221 119 SYCYHPDVMK 0.400 1222 228 LMNPVIYSVK 0.400 1223 73 RYAMILTDSR 0.240 1224 156 ILLSYVLIIR 0.240 1225 84 AQIGVASVIR 0.180 1226 64 RFVAVSNPLR 0.180 1227 230 NPVIYSVKTK 0.150 1228 233 IYSVKTKQIR 0.080 1229 50 TVMESSVLLA 0.080 1230 23 MLSIFWFNVR 0.080 1231 167 VLSVASPEER 0.080 1232 126 VMKLSCTDTR 0.080 1233 20 LVTMLSIFWF 0.060 1234 87 GVASVIRGLL 0.060 1235 183 CVSHIVAFAI 0.060 1236 239 KQIRRAVIKI 0.054 1237 65 FVAVSNPLRY 0.040 1238 101 MVALLIRLSY 0.040 1239 67 AVSNPLRYAM 0.040 1240 215 HTMIANTYLL 0.040 1241 148 TVGVDLLLIL 0.040 1242 94 GLLMLTPMVA 0.036 1243 168 LSVASPEERK 0.030 1244 147 STVGVDLLLI 0.030 1245 237 KTKQIRRAVI 0.030 1246 114 QVLHHSYCYH 0.030 1247 42 HMFFIKFFTV 0.024 1248 135 RINSAVGLTA 0.024 1249 220 NTYLLISPLM 0.020 1250 213 YVHTMIANTY 0.020 1251 238 TKQIRRAVIK 0.020 1252 113 SQVLHHSYCY 0.018 1253 22 TMLSIFWFNV 0.018 1254 208 KQAPAYVHTM 0.018 1255 191 AIYYIPLISL 0.016 1256 1 MYYFLSMLSA 0.016 1257 18 STLVTMLSIF 0.015 1258 49 FTVMESSVLL 0.015 1259 234 YSVKTKQIRR 0.012 1260 196 PLISLSIVHR 0.012 1261 58 LAMAFDRFVA 0.012 1262 150 GVDLLLILLS 0.012 1263 216 TMIANTYLLI 0.012 1264 193 YYIPLISLSI 0.012 1265 155 LILLSYVLII 0.012 1266 107 RLSYCHSQVL 0.012 1267 186 HIVAFAIYYI 0.012 1268 82 RIAQIGVASV 0.012 1269 47 KFFTVMESSV 0.012 1270 19 TLVTMLSIFW 0.012 1271 153 LLLILLSYVL 0.012 1272 14 GLSISTLVTM 0.012 1273 6 SMLSATDLGL 0.012 1274 161 VLIIRTVLSV 0.012 1275 103 ALLIRLSYCH 0.012 1276 131 CTDTRINSAV 0.010 1277 142 LTAMFSTVGV 0.010 1278 181 STCVSHIVAF 0.010 1279 109 SYCHSQVLHH 0.008 1280 144 AMFSTVGVDL 0.008 1281 39 CLSHMFFIKF 0.008 1282 97 MLTPMVALLI 0.008 1283 120 YCYHPDVMKL 0.008 1284 51 VMESSVLLAM 0.008 1285 95 LLMLTPMVAL 0.008 1286 188 VAFAIYYIPL 0.008 1287 99 TPMVALLIRL 0.008 1288 223 LLISPLMNPV 0.006 1289 159 SYVLIIRTVL 0.006 1290 154 LLILLSYVLI 0.006 1291 162 LIIRTVLSVA 0.006 1292 37 NACLSHMFFI 0.006 1293 205 RFGKQAPAYV 0.006 1294 178 ETFSTCVSHI 0.006 1295 139 AVGLTAMFST 0.006 1296 90 SVIRGLLMLT 0.006 1297 56 VLLAMAFDRF 0.006 1298 96 LMLTPMVALL 0.006 1299 195 IPLISLSIVH 0.006 1300 165 RTVLSVASPE 0.005 1301 21 VTMLSIFWFN 0.004 1302 197 LISLSIVHRF 0.004 1303 232 VIYSVKTKQI 0.004 1304 74 YAMILTDSRI 0.004 1305 43 MFFIKFFTVM 0.004 1306 187 IVAFAIYYIP 0.004 1307 4 FLSMLSATDL 0.004 1308 77 ILTDSRIAQI 0.004 1309 194 YIPLISLSIV 0.004 1310 224 LISPLMNPVI 0.004 1311 210 APAYVHTMIA 0.004 1312 88 VASVIRGLLM 0.004 1313 59 AMAFDRFVAV 0.004 1314 201 SIVHRFGKQA 0.003 1315 160 YVLIIRTVLS 0.003 1316

TABLE XII V1B-A11-10mers SEQ Pos 1234567890 Score ID 28 HTWISIPFCF 0.060 1317 9 ITSTSIIFLL 0.030 1318 39 SVTALLGNSL 0.020 1319 20 GVPGLEAFHT 0.018 1320 42 ALLGNSLILF 0.012 1321 40 VTALLGNSLI 0.010 1323 54 ITQPSLHEPM 0.010 1323 18 LTGVPGLEAF 0.010 1324 8 NITSTSIIFL 0.008 1325 58 SLHEPMYYFL 0.008 1326 17 LLTGVPGLEA 0.008 1327 43 LLGNSLILFA 0.008 1328 55 TQPSLHEPMY 0.006 1329 6 LQNITSTSII 0.006 1330 41 TALLGNSLIL 0.006 1331 33 IPFCFLSVTA 0.004 1333 56 QPSLHEPMYY 0.004 1333 26 AFHTWISIPF 0.004 1334 5 TLQNITSTSI 0.004 1335 35 FCFLSVTALL 0.004 1336 15 IFLLTGVPGL 0.003 1337 52 ATITQPSLHE 0.003 1338 23 GLEAFHTWIS 0.002 1339 50 LFATITQPSL 0.002 1340 51 FATITQPSLH 0.002 1341 11 STSIIFLLTG 0.002 1342 2 ITSTLQNITS 0.002 1343 21 VPGLEAFHTW 0.002 1344 4 STLQNITSTS 0.002 1345 47 SLILFATITQ 0.001 1346 7 QNITSTSIIF 0.001 1347 30 WISIPFCFLS 0.001 1348 45 GNSLILFATI 0.001 1349 24 LEAFHTWISI 0.001 1350 60 HEPMYYFLSM 0.001 1351 61 EPMYYFLSML 0.001 1352 49 ILFATITQPS 0.001 1353 14 IIFLLTGVPG 0.001 1354 37 FLSVTALLGN 0.001 1355 48 LILFATITQP 0.001 1356 36 CFLSVTALLG 0.001 1357 16 FLLTGVPGLE 0.001 1358 31 ISIPFCFLSV 0.001 1359 13 SIIFLLTGVP 0.001 1360 1 FITSTLQNIT 0.000 1361 32 SIPFCFLSVT 0.000 1362 53 TITQPSLHEP 0.000 1363 29 TWISIPFCFL 0.000 1364 19 TGVPGLEAFH 0.000 1365 12 TSIIFLLTGV 0.000 1366 34 PFCFLSVTAL 0.000 1367 62 PMYYFLSMLS 0.000 1368 25 EAFHTWISIP 0.000 1369 57 PSLHEPMYYF 0.000 1370 44 LGNSLILFAT 0.000 1371 59 LHEPMYYFLS 0.000 1372 10 TSTSIIFLLT 0.000 1373 22 PGLEAFHTWI 0.000 1374 38 LSVTALLGNS 0.000 1375 46 NSLILFATIT 0.000 1376 27 FHTWISIPFC 0.000 1377 3 TSTLQNITST 0.000 1378 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE XII V2-A11-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 5 NTYLLTSPLM 0.020 1379 9 LTSPLMNPVI 0.010 1380 8 LLTSPLMNPV 0.004 1381 7 YLLTSPLMNP 0.001 1382 6 TYLLTSPLMN 0:001 1383 1 TMIANTYLLT 0.001 1384 2 MIANTYLLTS 0.001 1385 4 ANTYLLTSPL 0.000 1386 3 IANTYLLTSP 0.000 1387 10 TSPLMNPVIY 0.000 1388

TABLE XIII V1-A24-9-mers: 238P1B2 SEQ Pos 123456789 Score ID 121 CYHPDVMKL 264.000 1389 192 IYYIPLISL 200.000 1390 64 RFVAVSNPL 100.800 1391 221 TYLLISPLM 52.500 1392 233 IYSVKTKQI 50.000 1393 145 MFSTVGVDL 28.000 1394 119 SYCYHPDVM 25.000 1395 189 AFAIYYIPL 20.000 1396 28 WFNVREISF 15.000 1397 73 RYAMILTDS 14.000 1398 193 YYIPLISLS 10.800 1399 159 SYVLIIRTV 10.500 1400 149 VGVDLLLIL 8.640 1401 147 STVGVDLLL 8.400 1402 160 YVLIIRTVL 8.400 1403 212 AYVHTMIAN 7.500 1404 154 LLILLSYVL 7.200 1405 50 TVMESSVLL 7.200 1406 96 LMLTPMVAL 6.000 1407 5 LSMLSATDL 6.000 1408 216 TMIANTYLL 6.000 1409 49 FTVMESSVL 6.000 1410 70 NPLRYAMIL 6.000 1411 1 MYYFLSMLS 6.000 1412 215 HTMIANTYL 6.000 1413 90 SVIRGLLML 6.000 1414 88 VASVIRGLL 5.600 1415 87 GVASVIRGL 5.600 1416 26 IFWFNVREI 5.500 1417 179 TFSTCVSHI 5.000 1418 2 YYFLSMLSA 5.000 1419 16 SISTLVTML 4.800 1420 108 LSYCHSQVL 4.800 1421 97 MLTPMVALL 4.800 1422 150 GVDLLLILL 4.800 1423 220 NTYLLISPL 4.800 1424 44 FFIKFFTVM 4.500 1425 198 ISLSIVHRF 4.200 1426 146 FSTVGVDLL 4.000 1427 7 MLSATDLGL 4.000 1428 12 DLGLSISTL 4.000 1429 35 SFNACLSHM 3.750 1430 138 SAVGLTAMF 3.600 1431 172 SPEERKETF 3.600 1432 54 SSVLLAMAF 3.600 1433 21 VTMLSIFWF 3.000 1434 182 TCVSHIVAF 3.000 1435 19 TLVTMLSIF 3.000 1436 40 LSHMFFIKF 2.640 1437 57 LLAMAFDRF 2.400 1438 36 FNACLSHMF 2.400 1439 98 LTPMVALLI 2.100 1440 194 YIPLISLSI 2.100 1441 84 AQIGVASVI 2.100 1442 128 KLSCTDTRI 2.000 1443 37 NACLSHMFF 2.000 1444 190 FAIYYIPLI 1.500 1445 156 ILLSYVLII 1.500 1446 69 SNPLRYAMI 1.500 1447 75 AMILTDSRI 1.500 1448 225 ISPLMNPVI 1.500 1449 18 STLVTMLSI 1.500 1450 155 LILLSYVLI 1.500 1451 93 RGLLMLTPM 1.500 1452 38 ACLSHMFFI 1.500 1453 9 SATDLGLSI 1.440 1454 47 KFFTVMESS 1.400 1455 210 APAYVHTMI 1.400 1456 184 VSHIVAFAI 1.400 1457 32 REISFNACL 1.200 1458 240 QIRRAVIKI 1.100 1459 68 VSNPLRYAM 1.080 1460 205 RFGKQAPAY 1.000 1461 217 MIANTYLLI 1.000 1462 187 IVAFAIYYI 1.000 1463 148 TVGVDLLLI 1.000 1464 78 LTDSRIAQI 1.000 1465 89 ASVIRGLLM 0.750 1466 3 YFLSMLSAT 0.750 1467 209 QAPAYVHTM 0.750 1468 15 LSISTLVTM 0.750 1469 100 PMVALLIRL 0.720 1470 134 TRINSAVGL 0.600 1471 43 MFFIKFFTV 0.600 1472 241 IRRAVIKIL 0.560 1473 48 FFTVMESSV 0.500 1474 137 NSAVGLTAM 0.500 1475 61 AFDRFVAVS 0.500 1476 109 SYCHSQVLH 0.500 1477 243 RAVIKILHS 0.300 1478 41 SHMFFIKFF 0.300 1479 135 RINSAVGLT 0.300 1480 237 KTKQIRRAV 0.280 1481 82 RIAQIGVAS 0.280 1482 22 TMLSIFWFN 0.252 1483 228 LMNPVIYSV 0.252 1484 208 KQAPAYVHT 0.240 1485 152 DLLLILLSY 0.210 1486 13 LGLSISTLV 0.210 1487 102 VALLIRLSY 0.210 1488

TABLE XIII V1B-A24-9mers: 238P1B2 SEQ Pos 123456789 Score ID 36 CFLSVTALL 42.000 1489 42 ALLGNSLIL 6.000 1490 16 FLLTGVPGL 6.000 1491 40 VTALLGNSL 5.760 1492 10 TSTSIIFLL 5.600 1493 30 WISIPFCFL 4.800 1494 29 TWISIPFCF 4.200 1495 51 FATITQPSL 4.000 1496 9 ITSTSIIFL 4.000 1497 35 FCFLSVTAL 4.000 1498 19 TGVPGLEAF 3.600 1499 58 SLHEPMYYF 2.400 1500 46 NSLILFATI 2.160 1501 43 LLGNSLILF 2.000 1502 8 NITSTSIIF 2.000 1503 7 QNITSTSII 1.500 1504 41 TALLGNSLI 1.500 1505 6 LQNITSTSI 1.500 1506 23 GLEAFHTWI 1.500 1507 1 FITSTLQNI 1.200 1508 25 EAFHTWISI 1.000 1509 61 EPMYYFLSM 0.900 1510 55 TQPSLHEPM 0.900 1511 50 LFATITQPS 0.840 1512 59 LHEPMYYFL 0.720 1513 62 PMYYFLSML 0.400 1514 27 FHTWISIPF 0.280 1515 5 TLQNITSTS 0.210 1516 31 ISIPFCFLS 0.180 1517 44 LGNSLILFA 0.180 1518 13 STIFLLTGV 0.180 1519 32 SIPFCFLSV 0.180 1520 11 STSIIFLLT 0.168 1521 47 SLILFATIT 0.150 1522 4 STLQNITST 0.150 1523 38 LSVTALLGN 0.150 1524 2 ITSTLQNIT 0.144 1525 45 GNSLILFAT 0.140 1526 39 SVTALLGNS 0.120 1527 18 LTGVPGLEA 0.110 1528 56 QPSLHEPMY 0.100 1529 28 HTWISIPFC 0.100 1530 3 TSTLQNITS 0.100 1531 33 IPFCFLSVT 0.100 1532 21 VPGLEAFHT 0.100 1533 15 IFLLTGVPG 0.075 1534 34 PFCFLSVTA 0.060 1535 26 AFHTWISIP 0.050 1536 22 PGLEAFHTW 0.022 1537 54 ITQPSLHEP 0.020 1538 20 GVPGLEAFH 0.018 1539 12 TSIIFLLTG 0.015 1540 48 LILFATITQ 0.015 1541 57 PSLHEPMYY 0.015 1542 52 ATITQPSLH 0.015 1543 60 HEPMYYFLS 0.015 1544 17 LLTGVPGLE 0.014 1545 14 IIFLLTGVP 0.012 1546 24 LEAFHTWIS 0.010 1547 49 ILFATITQP 0.010 1548 37 FLSVTALLG 0.010 1549 53 TITQPSLHE 0.010 1550 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE XIII V2-A24-9mers SEQ Pos 123456789 Score ID 5 TYLLTSPLM 37.500 1551 4 NTYLLTSPL 4.800 1552 9 TSPLMNPVI 1.500 1553 8 LTSPLMNPV 0.173 1554 6 YLLTSPLMN 0.150 1555 2 IANTYLLTS 0.150 1556 1 MIANTYLLT 0.100 1557 3 ANTYLLTSP 0.012 1558 7 LLTSPLMNP 0.010 1559

TABLE XIV V1-A24-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 159 SYVLIIRTVL 420.000 1560 193 YYIPLISLSI 126.000 1561 145 MFSTVGVDLL 20.000 1562 48 FFTVMESSVL 20.000 1563 35 SFNACLSHMF 18.000 1564 107 RLSYCHSQVL 9.600 1565 149 VGVDLLLILL 8.640 1566 121 CYHPDVMKLS 8.400 1567 86 IGVASVIRGL 8.400 1568 221 TYLLISPLMN 7.500 1569 212 AYVHTMIANT 7.500 1570 99 TPMVALLIRL 7.200 1571 15 LSISTLVTML 7.200 1572 153 LLLILLSYVL 7.200 1573 6 SMLSATDLGL 6.000 1574 49 FTVMESSVLL 6.000 1575 69 SNPLRYAMIL 6.000 1576 96 LMLTPMVALL 6.000 1577 89 ASVIRGLLML 6.000 1578 95 LLMLTPMVAL 6.000 1579 192 IYYIPLISLS 6.000 1580 215 HTMIANTYLL 6.000 1581 144 AMFSTVGVDL 5.600 1582 146 FSTVGVDLLL 5.600 1583 240 QIRRAVIKIL 5.600 1584 87 GVASVIRGLL 5.600 1585 2 YYFLSMLSAT 5.000 1586 1 MYYFLSMLSA 5.000 1587 189 AFAIYYIPLI 5.000 1588 219 ANTYLLISPL 4.800 1589 148 TVGVDLLLIL 4.800 1590 120 YCYHPDVMKL 4.400 1591 133 DTRINSAVGL 4.000 1592 188 VAFAIYYIPL 4.000 1593 4 FLSMLSATDL 4.000 1594 191 AIYYIPLISL 4.000 1595 56 VLLAMAFDRF 3.600 1596 171 ASPEERKETF 3.600 1597 239 KQIRRAVIKI 3.300 1598 43 MFFIKFFTVM 3.000 1599 18 STLVTMLSIF 3.000 1600 197 LISLSIVHRF 2.800 1601 39 CLSHMFFIKF 2.640 1602 53 ESSVLLAMAF 2.400 1603 137 NSAVGLTAMF 2.400 1604 209 QAPAYVHTMI 2.100 1605 83 IAQIGVASVI 2.100 1606 237 KTKQIRRAVI 2.000 1607 36 FNACLSHMFF 2.000 1608 181 STCVSHIVAF 2.000 1609 27 FWFNVREISF 2.000 1610 40 LSHMFFIKFF 2.000 1611 20 LVTMLSIFWF 2.000 1612 68 VSNPLRYAMI 1.800 1613 97 MLTPMVALLI 1.680 1614 74 YAMILTDSRI 1.500 1615 154 LLILLSYVLI 1.500 1616 155 LILLSYVLII 1.500 1617 186 HIVAFAIYYI 1.500 1618 147 STVGVDLLLI 1.500 1619 216 TMIANTYLLI 1.500 1620 183 CVSHIVAFAI 1.400 1621 224 LISPLMNPVI 1.200 1622 77 ILTDSRIAQI 1.200 1623 8 LSATDLGLSI 1.200 1624 208 KQAPAYVHTM 1.200 1625 73 RYAMILTDSR 1.200 1626 25 SIFWFNVREI 1.100 1627 51 VMESSVLLAM 1.050 1628 232 VIYSVKTKQI 1.000 1629 178 ETFSTCVSHI 1.000 1630 37 NACLSHMFFI 1.000 1631 17 ISTLVTMLSI 1.000 1632 205 RFGKQAPAYV 1.000 1633 47 KFFTVMESSV 1.000 1634 28 WFNVREISFN 0.750 1635 26 IFWFNVREIS 0.700 1636 220 NTYLLISPLM 0.700 1637 61 AFDRFVAVSN 0.700 1638 179 TFSTCVSHIV 0.700 1639 63 DRFVAVSNPL 0.672 1640 11 TDLGLSISTL 0.600 1641 67 AVSNPLRYAM 0.600 1642 233 IYSVKTKQIR 0.600 1643 31 VREISFNACL 0.600 1644 14 GLSISTLVTM 0.500 1645 88 VASVIRGLLM 0.500 1646 118 HSYCYHPDVM 0.500 1647 119 SYCYHPDVMK 0.500 1648 34 ISFNACLSHM 0.500 1649 109 SYCHSQVLHH 0.500 1650 136 INSAVGLTAM 0.500 1651 135 RINSAVGLTA 0.420 1652 214 VHTMIANTYL 0.400 1653 93 RGLLMLTPMV 0.360 1654 21 VTMLSIFWFN 0.252 1655 223 LLISPLMNPV 0.216 1656 190 FAIYYIPLIS 0.210 1657 162 LIIRTVLSVA 0.210 1658 29 FNVREISFNA 0.210 1659

TABLE XIV V1B-A24-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 15 IFLLTGVPGL 30.000 1660 50 LFATITQPSL 20.000 1661 26 AFHTWISTPF 14.000 1662 29 TWISIPFCFL 7.200 1663 41 TALLGNSLIL 6.000 1664 61 EPMYYFLSML 6.000 1665 39 SVTALLGNSL 5.760 1666 35 FCFLSVTALL 5.600 1667 9 ITSTSIIFLL 5.600 1668 58 SLHEPMYYFL 4.800 1669 8 NITSTSIIFL 4.000 1670 7 QNITSTSIIF 3.000 1671 42 ALLGNSLILF 3.000 1672 28 HTWISIPFCF 2.800 1673 34 PFCFLSVTAL 2.000 1674 18 LTGVPGLEAF 2.000 1675 5 TLQNITSTSI 1.500 1676 6 LQNITSTSII 1.500 1677 45 GNSLILFATT 1.440 1678 54 ITQPSLHEPM 1.080 1679 40 VTALLGNSLI 1.000 1680 57 PSLHEPMYYF 0.300 1681 31 ISIPFCFLSV 0.216 1682 4 STLQNITSTS 0.210 1683 44 LGNSLILFAT 0.210 1684 22 PGLEAFHTWI 0.180 1685 12 TSIIFLLTGV 0.180 1686 38 LSVTALLGNS 0.180 1687 10 TSTSIIFLLT 0.168 1688 49 ILFATITQPS 0.168 1689 20 GVPGLEAFHT 0.150 1690 32 SIPFCFLSVT 0.150 1691 46 NSLILFATIT 0.150 1692 23 GLEAFHTWIS 0.150 1693 55 TQPSLHEPMY 0.150 1694 1 FITSTLQNIT 0.144 1695 43 LLGNSLILFA 0.120 1696 33 IPFCFLSVTA 0.120 1697 21 VPGLEAFHTW 0.120 1698 17 LLTGVPGLEA 0.110 1699 3 TSTLQNITST 0.100 1700 56 QPSLHEPMYY 0.100 1701 24 LEAFHTWISI 0.100 1702 30 WISIPFCFLS 0.100 1703 37 FLSVTALLGN 0.100 1704 2 ITSTLQNITS 0.100 1705 60 HEPMYYFLSM 0.090 1706 36 CFLSVTALLG 0.075 1707 19 TGVPGLEAFH 0.022 1708 16 FLLTGVPGLE 0.021 1709 59 LHEPMYYFLS 0.018 1710 13 SIIFLLTGVP 0.018 1711 48 LILFATITQP 0.015 1712 52 ATITQPSLHE 0.015 1713 47 SLILFATITQ 0.015 1714 62 PMYYFLSMLS 0.012 1715 53 TITQPSLHEP 0.011 1716 14 IIFLLTGVPG 0.010 1717 27 FHTWISIPFC 0.010 1718 11 STSIIFLLTG 0.010 1719 25 EAFHTWISIP 0.010 1720 51 FATITQPSLH 0.010 1721 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE XIV V2-A24-10mers SEQ Pos 1234567890 Score ID 6 TYLLTSPLMN 7.500 1722 4 ANTYLLTSPL 4.800 1723 9 LTSPLMNPVI 1.200 1724 5 NTYLLTSPLM 0.500 1725 1 TMIANTYLLT 0.150 1726 10 TSPLMNPVIY 0.150 1727 8 LLTSPLMNPV 0.144 1728 2 MIANTYLLTS 0.100 1729 3 IANTYLLTSP 0.018 1730 7 YLLTSPLMNP 0.015 1731

TABLE XV V1-B7-9-mers: 238P1B2 SEQ Pos 123456789 Score ID 70 NPLRYAMIL 80.000 1732 50 TVMESSVLL 60.000 1733 160 YVLIIRTVL 30.000 1734 210 APAYVHTMI 24.000 1735 90 SVIRGLLML 20.000 1736 87 GVASVIRGL 20.000 1737 88 VASVIRGLL 18.000 1738 215 HTMIANTYL 12.000 1739 5 LSMLSATDL 12.000 1740 150 GVDLLLILL 6.000 1741 96 LMLTPMVAL 6.000 1742 30 NVREISFNA 5.000 1743 108 LSYCHSQVL 4.000 1744 16 SISTLVTML 4.000 1745 154 LLILLSYVL 4.000 1746 241 IRRAVIKIL 4.000 1747 146 FSTVGVDLL 4.000 1748 149 VGVDLLLIL 4.000 1749 240 QIRRAVIKI 4.000 1750 216 TMIANTYLL 4.000 1751 220 NTYLLISPL 4.000 1752 49 FTVMESSVL 4.000 1753 195 IPLISLSIV 4.000 1754 7 MLSATDLGL 4.000 1755 147 STVGVDLLL 4.000 1756 12 DLGLSISTL 4.000 1757 97 MLTPMVALL 4.000 1758 89 ASVIRGLLM 3.000 1759 209 QAPAYVHTM 3.000 1760 67 AVSNPLRYA 2.250 1761 148 TVGVDLLLI 2.000 1762 80 DSRIAQIGV 2.000 1763 230 NPVIYSVKT 2.000 1764 187 IVAFAIYYI 2.000 1765 58 LAMAFDRFV 1.800 1766 143 TAMFSTVGV 1.800 1767 68 VSNPLRYAM 1.500 1768 189 AFAIYYIPL 1.200 1769 9 SATDLGLSI 1.200 1770 75 AMILTDSRI 1.200 1771 190 FAIYYIPLI 1.200 1772 38 ACLSHMFFI 1.200 1773 84 AQIGVASVI 1.200 1774 93 RGLLMLTPM 1.000 1775 163 IIRTVLSVA 1.000 1776 91 VIRGLLMLT 1.000 1777 15 LSISTLVTM 1.000 1778 137 NSAVGLTAM 1.000 1779 60 MAFDRFVAV 0.600 1780 123 HPDVMKLSC 0.600 1781 99 TPMVALLIR 0.600 1782 83 IAQIGVASV 0.600 1783 192 IYYIPLISL 0.600 1784 213 YVHTMIANT 0.500 1785 202 IVHRFGKQA 0.500 1786 183 CVSHIVAFA 0.500 1787 171 ASPEERKET 0.450 1788 59 AMAFDRFVA 0.450 1789 64 RFVAVSNPL 0.400 1790 69 SNPLRYAMI 0.400 1791 98 LTPMVALLI 0.400 1792 121 CYHPDVMKL 0.400 1793 32 REISFNACL 0.400 1794 18 STLVTMLSI 0.400 1795 217 MIANTYLLI 0.400 1796 145 MFSTVGVDL 0.400 1797 100 PMVALLIRL 0.400 1798 128 KLSCTDTRI 0.400 1799 184 VSHIVAFAI 0.400 1800 194 YIPLISLSI 0.400 1801 155 LILLSYVLI 0.400 1802 134 TRINSAVGL 0.400 1803 225 ISPLMNPVI 0.400 1804 156 ILLSYVLII 0.400 1805 226 SPLMNPVIY 0.400 1806 237 KTKQIRRAV 0.300 1807 103 ALLIRLSYC 0.300 1808 139 AVGLTAMFS 0.300 1809 95 LLMLTPMVA 0.300 1810 13 LGLSISTLV 0.200 1811 105 LIRLSYCHS 0.200 1812 23 MLSIFWFNV 0.200 1813 162 LIIRTVLSV 0.200 1814 153 LLLILLSYV 0.200 1815 206 FGKQAPAYV 0.200 1816 94 GLLMLTPMV 0.200 1817 180 FSTCVSHIV 0.200 1818 107 RLSYCHSQV 0.200 1819 228 LMNPVIYSV 0.200 1820 141 GLTAMFSTV 0.200 1821 224 LISPLMNPV 0.200 1822 118 HSYCYHPDV 0.200 1823 125 DVMKLSCTD 0.150 1824 244 AVIKILHSK 0.150 1825 172 SPEERKETF 0.120 1826 78 LTDSRIAQI 0.120 1827 52 MESSVLLAM 0.100 1828 14 GLSISTLVT 0.100 1829 158 LSYVLIIRT 0.100 1830 119 SYCYHPDVM 0.100 1831

TABLE XV V1B-B7-9mers: 238P1B2 SEQ Pos 123456789 Score ID 61 EPMYYFLSM 60.000 1832 51 FATITQPSL 12.000 1833 42 ALLGNSLIL 12.000 1834 30 WISIPFCFL 6.000 1835 16 FLLTGVPGL 4.000 1836 10 TSTSIIFLL 4.000 1837 40 VTALLGNSL 4.000 1838 35 FCFLSVTAL 4.000 1839 9 ITSTSIIFL 4.000 1840 21 VPGLEAFHT 2.000 1841 33 IPFCFLSVT 2.000 1842 25 EAFHTWISI 1.200 1843 41 TALLGNSLI 1.200 1844 55 TQPSLHEPM 1.000 1845 36 CFLSVTALL 0.400 1846 7 QNITSTSII 0.400 1847 1 FITSTLQNI 0.400 1848 46 NSLILFATI 0.400 1849 56 QPSLHEPMY 0.400 1850 6 LQNITSTSI 0.400 1851 62 PMYYFLSML 0.400 1852 13 SIIFLLTGV 0.200 1853 32 SIPFCFLSV 0.200 1854 18 LTGVPGLEA 0.150 1855 59 LHEPMYYFL 0.120 1856 23 GLEAFHTWI 0.120 1857 45 GNSLILFAT 0.100 1858 47 SLILFATIT 0.100 1859 44 LGNSLILFA 0.100 1860 4 STLQNITST 0.100 1861 11 STSIIFLLT 0.100 1862 2 ITSTLQNIT 0.100 1863 39 SVTALLGNS 0.100 1864 28 HTWISIPFC 0.100 1865 20 GVPGLEAFH 0.050 1866 58 SLHEPMYYF 0.030 1867 52 ATITQPSLH 0.030 1868 38 LSVTALLGN 0.020 1869 5 TLQNITSTS 0.020 1870 43 LLGNSLILF 0.020 1871 31 ISIPFCFLS 0.020 1872 19 TGVPGLEAF 0.020 1873 3 TSTLQNITS 0.020 1874 8 NITSTSIIF 0.020 1875 53 TITQPSLHE 0.015 1876 54 ITQPSLHEP 0.010 1877 14 IIFLLTGVP 0.010 1878 37 FLSVTALLG 0.010 1879 49 TLFATITQP 0.010 1880 48 LILFATITQ 0.010 1881 12 TSIIFLLTG 0.010 1882 17 LLTGVPGLE 0.010 1883 26 AFHTWISIP 0.003 1884 29 TWISIPFCF 0.002 1885 50 LFATITQPS 0.002 1886 27 FHTWISIPF 0.002 1887 22 PGLEAFHTW 0.002 1888 24 LEAFHTWIS 0.002 1889 60 HEPMYYFLS 0.002 1890 57 PSLHEPMYY 0.002 1891 15 IFLLTGVPG 0.001 1892 34 PFCFLSVTA 0.001 1893 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE XV V2-B7-9mers-238P1B2 SEQ Pos 123456789 Score ID 4 NTYLLTSPL 4.000 1894 9 TSPLMNPVI 0.400 1895 8 LTSPLMNPV 0.200 1896 5 TYLLTSPLM 0.100 1897 1 MIANTYLLT 0.100 1898 2 IANTYLLTS 0.060 1899 3 ANTYLLTSP 0.030 1900 6 YLLTSPLMN 0.020 1901 7 LLTSPLMNP 0.015 1902

TABLE XVI V1-B7-10-mers: 238P182 SEQ Pos 1234567890 Score ID 99 TPMVALLIRL 240.000 1903 133 DTRINSAVGL 40.000 1904 240 QIRRAVIKIL 40.000 1905 87 GVASVIRGLL 30.000 1906 67 AVSNPLRYAM 22.500 1907 148 TVGVDLLLIL 20.000 1908 191 AIYYIPLISL 18.000 1909 95 LLMLTPMVAL 18.000 1910 219 ANTYLLISPL 12.000 1911 144 AMFSTVGVDL 12.000 1912 89 ASVIRGLLML 12.000 1913 215 HTMIANTYLL 12.000 1914 188 VAFAIYYIPL 12.000 1915 210 APAYVHTMIA 6.000 1916 30 NVREISFNAC 5.000 1917 86 IGVASVIRGL 4.000 1918 107 RLSYCHSQVL 4.000 1919 149 VGVDLLLILL 4.000 1920 146 FSTVGVDLLL 4.000 1921 69 SNPLRYAMIL 4.000 1922 96 LMLTPMVALL 4.000 1923 49 FTVMESSVLL 4.000 1924 6 SMLSATDLGL 4.000 1925 4 FLSMLSATDL 4.000 1926 120 YCYHPDVMKL 4.000 1927 15 LSISTLVTML 4.000 1928 153 LLLILLSYVL 4.000 1929 74 YAMILTDSRI 3.600 1930 88 VASVIRGLLM 3.000 1931 183 CVSHIVAFAI 2.000 1932 70 NPLRYAMILT 2.000 1933 139 AVGLTAMFST 1.500 1934 125 DVMKLSCTDT 1.500 1935 50 TVMESSVLLA 1.500 1936 58 LAMAFDRFVA 1.350 1937 209 QAPAYVHTMI 1.200 1938 37 NACLSHMFFI 1.200 1939 83 IAQIGVASVI 1.200 1940 80 DSRIAQIGVA 1.000 1941 220 NTYLLISPLM 1.000 1942 34 ISFNACLSHM 1.000 1943 118 HSYCYHPDVM 1.000 1944 136 INSAVGLTAM 1.000 1945 208 KQAPAYVHTM 1.000 1946 14 GLSISTLVTM 1.000 1947 59 AMAFDRFVAV 0.600 1948 237 KTKQIRRAVI 0.600 1949 226 SPLMNPVIYS 0.600 1950 123 HPDVMKLSCT 0.600 1951 159 SYVLIIRTVL 0.600 1952 90 SVIRGLLMLT 0.500 1953 235 SVKTKQIRRA 0.500 1954 66 VAVSNPLRYA 0.450 1955 170 VASPEERKET 0.450 1956 224 LISPLMNPVI 0.400 1957 48 FFTVMESSVL 0.400 1958 214 VHTMIANTYL 0.400 1959 154 LLILLSYVLI 0.400 1960 25 SIFWFNVREI 0.400 1961 8 LSATDLGLSI 0.400 1962 232 VIYSVKTKQI 0.400 1963 11 TDLGLSISTL 0.400 1964 97 MLTPMVALLI 0.400 1965 17 ISTLVTMLSI 0.400 1966 186 HIVAFAIYYI 0.400 1967 239 KQIRRAVIKI 0.400 1968 216 TMIANTYLLI 0.400 1969 145 MFSTVGVDLL 0.400 1970 68 VSNPLRYAMI 0.400 1971 77 ILTDSRIAQI 0.400 1972 147 STVGVDLLLI 0.400 1973 63 DRFVAVSNPL 0.400 1974 155 LILLSYVLII 0.400 1975 178 ETFSTCVSHI 0.400 1976 75 AMILTDSRIA 0.300 1977 51 VMESSVLLAM 0.300 1978 102 VALLIRLSYC 0.300 1979 223 LLISPLMNPV 0.200 1980 195 IPLISLSIVH 0.200 1981 161 VLIIRTVLSV 0.200 1982 142 LTAMFSTVGV 0.200 1983 194 YIPLISLSIV 0.200 1984 230 NPVIYSVKTK 0.200 1985 93 RGLLMLTPMV 0.200 1986 57 LLAMAFDRFV 0.200 1987 152 DLLLILLSYV 0.200 1988 140 VGLTAMFSTV 0.200 1989 158 LSYVLIIRTV 0.200 1990 42 HMFFIKFFTV 0.200 1991 82 RIAQIGVASV 0.200 1992 163 IIRTVLSVAS 0.200 1993 22 TMLSIFWFNV 0.200 1994 12 DLGLSISTLV 0.200 1995 101 MVALLIRLSY 0.150 1996 244 AVIKILHSKE 0.150 1997 172 SPEERKETFS 0.120 1998 189 AFAIYYIPLI 0.120 1999 31 VREISFNACL 0.120 2000 92 IRGLLMLTPM 0.100 2001 160 YVLIIRTVLS 0.100 2002

TABLE XVI V1B-B7-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 61 EPMYYFLSML 240.000 2003 39 SVTALLGNSL 20.000 2004 41 TALLGNSLIL 12.000 2005 35 FCFLSVTALL 4.000 2006 58 SLHEPMYYFL 4.000 2007 9 ITSTSIIFLL 4.000 2008 8 NITSTSIIFL 4.000 2009 33 IPFCFLSVTA 2.000 2010 54 ITQPSLHEPM 1.000 2011 29 TWISIPFCFL 0.600 2012 20 GVPGLEAFHT 0.500 2013 50 LFATITQPSL 0.400 2014 56 QPSLHEPMYY 0.400 2015 40 VTALLGNSLI 0.400 2016 6 LQNITSTSII 0.400 2017 45 GNSLILFATI 0.400 2018 15 IFLLTGVPGL 0.400 2019 5 TLQNITSTSI 0.400 2020 21 VPGLEAFHTW 0.400 2021 31 ISIPFCFLSV 0.200 2022 12 TSIIFLLTGV 0.200 2023 17 LLTGVPGLEA 0.150 2024 44 LGNSLILFAT 0.100 2025 32 SIPFCFLSVT 0.100 2026 10 TSTSIIFLLT 0.100 2027 3 TSTLQNITST 0.100 2028 43 LLGNSLILFA 0.100 2029 60 HEPMYYFLSM 0.100 2030 46 NSLILFATIT 0.100 2031 1 FITSTLQNIT 0.100 2032 42 ALLGNSLILF 0.060 2033 52 ATITQPSLHE 0.045 2034 34 PFCFLSVTAL 0.040 2035 22 PGLEAFHTWI 0.040 2036 24 LEAFHTWISI 0.040 2037 51 FATITQPSLH 0.030 2038 25 EAFHTWISIP 0.030 2039 18 LTGVPGLEAF 0.020 2040 30 WISIPFCFLS 0.020 2041 37 FLSVTALLGN 0.020 2042 38 LSVTALLGNS 0.020 2043 49 ILFATITQPS 0.020 2044 4 STLQNITSTS 0.020 2045 28 HTWISIPFCF 0.020 2046 7 QNITSTSIIF 0.020 2047 2 ITSTLQNITS 0.020 2048 55 TQPSLHEPMY 0.020 2049 19 TGVPGLEAFH 0.010 2050 48 LILFATITQP 0.010 2051 53 TITQPSLHEP 0.010 2052 14 IIFLLTGVPG 0.010 2053 13 SIIFLLTGVP 0.010 2054 47 SLILFATITQ 0.010 2055 11 STSIIFLLTG 0.010 2056 16 FLLTGVPGLE 0.010 2057 27 FHTWISIPFC 0.010 2058 26 AFHTWISIPF 0.006 2059 23 GLEAFHTWIS 0.006 2060 57 PSLHEPMYYF 0.003 2061 62 PMYYFLSMLS 0.002 2062 36 CFLSVTALLG 0.001 2063 59 LHEPMYYFLS 0.001 2064 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE XVI V2-B7-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 4 ANTYLLTSPL 12.000 2065 5 NTYLLTSPLM 1.000 2066 9 LTSPLMNPVI 0.400 2067 8 LLTSPLMNPV 0.200 2068 1 TMIANTYLLT 0.100 2069 3 IANTYLLTSP 0.030 2070 10 TSPLMNPVIY 0.020 2071 2 MIANTYLLTS 0.020 2072 7 YLLTSPLMNP 0.015 2073 6 TYLLTSPLMN 0.002 2074

TABLE XVII V1-B35-9-mers: 238P1B2 SEQ Pos 123456789 Score ID 226 SPLMNPVIY 40.000 2075 70 NPLRYAMIL 20.000 2076 172 SPEERKETF 12.000 2077 68 VSNPLRYAM 10.000 2078 89 ASVIRGLLM 10.000 2079 137 NSAVGLTAM 10.000 2080 112 HSQVLHHSY 10.000 2081 15 LSISTLVTM 10.000 2082 210 APAYVHTMI 8.000 2083 209 QAPAYVHTM 6.000 2084 102 VALLIRLSY 6.000 2085 66 VAVSNPLRY 6.000 2086 146 FSTVGVDLL 5.000 2087 5 LSMLSATDL 5.000 2088 54 SSVLLAMAF 5.000 2089 40 LSHMFFIKF 5.000 2090 108 LSYCHSQVL 5.000 2091 198 ISLSIVHRF 5.000 2092 195 IPLISLSIV 4.000 2093 93 RGLLMLTPM 4.000 2094 80 DSRIAQIGV 3.000 2095 88 VASVIRGLL 3.000 2096 37 NACLSHMFF 3.000 2097 138 SAVGLTAMF 3.000 2098 9 SATDLGLSI 2.400 2099 186 HIVAFAIYY 2.000 2100 225 ISPLMNPVI 2.000 2101 50 TVMESSVLL 2.000 2102 230 NPVIYSVKT 2.000 2103 152 DLLLILLSY 2.000 2104 114 QVLHHSYCY 2.000 2105 149 VGVDLLLIL 2.000 2106 184 VSHIVAFAI 2.000 2107 49 FTVMESSVL 1.500 2108 171 ASPEERKET 1.500 2109 60 MAFDRFVAV 1.200 2110 237 KTKQIRRAV 1.200 2111 240 QIRRAVIKI 1.200 2112 190 FAIYYIPLI 1.200 2113 118 HSYCYHPDV 1.000 2114 19 TLVTMLSIF 1.000 2115 57 LLAMAFDRF 1.000 2116 36 FNACLSHMF 1.000 2117 16 SISTLVTML 1.000 2118 216 TMIANTYLL 1.000 2119 12 DLGLSISTL 1.000 2120 182 TCVSHIVAF 1.000 2121 87 GVASVIRGL 1.000 2122 220 NTYLLISPL 1.000 2123 154 LLILLSYVL 1.000 2124 7 MLSATDLGL 1.000 2125 215 HTMIANTYL 1.000 2126 96 LMLTPMVAL 1.000 2127 97 MLTPMVALL 1.000 2128 160 YVLIIRTVL 1.000 2129 90 SVIRGLLML 1.000 2130 180 FSTCVSHIV 1.000 2131 21 VTMLSIFWF 1.000 2132 147 STVGVDLLL 1.000 2133 128 KLSCTDTRI 0.800 2134 129 LSCTDTRIN 0.750 2135 8 LSATDLGLS 0.750 2136 83 IAQIGVASV 0.600 2137 206 FGKQAPAYV 0.600 2138 243 RAVIKILHS 0.600 2139 148 TVGVDLLLI 0.600 2140 30 NVREISFNA 0.600 2141 123 HPDVMKLSC 0.600 2142 143 TAMFSTVGV 0.600 2143 58 LAMAFDRFV 0.600 2144 53 ESSVLLAMA 0.500 2145 17 ISTLVTMLS 0.500 2146 20 LVTMLSIFW 0.500 2147 158 LSYVLIIRT 0.500 2148 98 LTPMVALLI 0.400 2149 107 RLSYCHSQV 0.400 2150 217 MIANTYLLI 0.400 2151 187 IVAFAIYYI 0.400 2152 155 LILLSYVLI 0.400 2153 38 ACLSHMFFI 0.400 2154 205 RFGKQAPAY 0.400 2155 156 ILLSYVLII 0.400 2156 18 STLVTMLSI 0.400 2157 75 AMILTDSRI 0.400 2158 84 AQIGVASVI 0.400 2159 194 YIPLISLSI 0.400 2160 69 SNPLRYAMI 0.400 2161 126 VMKLSCTDT 0.300 2162 150 GVDLLLILL 0.300 2163 163 ITRTVLSVA 0.300 2164 91 VIRGLLMLT 0.300 2165 105 LTRLSYCHS 0.300 2166 241 IRRAVIKIL 0.300 2167 218 IANTYLLIS 0.300 2168 119 SYCYHPDVM 0.200 2169 228 LMNPVIYSV 0.200 2170 32 REISFNACL 0.200 2171 141 GLTAMFSTV 0.200 2172 162 LIIRTVLSV 0.200 2173 52 MESSVLLAM 0.200 2174

TABLE XVII V1B-B35-9mers: 238P1B2 SEQ Pos 123456789 Score ID 61 EPMYYFLSM 40.000 2175 56 QPSLHEPMY 40.000 2176 10 TSTSIIFLL 5.000 2177 21 VPGLEAFHT 3.000 2178 51 FATITQPSL 3.000 2179 46 NSLILFATI 2.000 2180 58 SLHEPMYYF 2.000 2181 33 IPFCFLSVT 2.000 2182 55 TQPSLHEPM 2.000 2183 57 PSLHEPMYY 1.500 2184 41 TALLGNSLI 1.200 2185 25 EAFHTWISI 1.200 2186 30 WISIPFCFL 1.000 2187 43 LLGNSLILF 1.000 2188 16 FLLTGVPGL 1.000 2189 19 TGVPGLEAF 1.000 2190 8 NITSTSIIF 1.000 2191 35 FCFLSVTAL 1.000 2192 42 ALLGNSLIL 1.000 2193 40 VTALLGNSL 1.000 2194 9 ITSTSIIFL 1.000 2195 31 ISIPFCFLS 0.500 2196 3 TSTLQNITS 0.500 2197 38 LSVTALLGN 0.500 2198 7 QNITSTSII 0.400 2199 6 LQNITSTSI 0.400 2200 1 FITSTLQNI 0.400 2201 32 SIPFCFLSV 0.200 2202 13 SIIFLLTGV 0.200 2203 23 GLEAFHTWI 0.120 2204 44 LGNSLILFA 0.100 2205 47 SLILFATIT 0.100 2206 5 TLQNITSTS 0.100 2207 4 STLQNITST 0.100 2208 22 PGLEAFHTW 0.100 2209 11 STSIIFLLT 0.100 2210 27 FHTWISIPF 0.100 2211 39 SVTALLGNS 0.100 2212 45 GNSLTLFAT 0.100 2213 18 LTGVPGLEA 0.100 2214 28 HTWISIPFC 0.100 2215 62 PMYYFLSML 0.100 2216 29 TWISIPFCF 0.100 2217 2 ITSTLQNIT 0.100 2218 36 CFLSVTALL 0.100 2219 12 TSIIFLLTG 0.050 2220 59 LHEPMYYFL 0.030 2221 54 ITQPSLHEP 0.010 2222 52 ATITQPSLH 0.010 2223 37 FLSVTALLG 0.010 2224 48 LILFATITQ 0.010 2225 14 ITFLLTGVP 0.010 2226 20 GVPGLEAFH 0.010 2227 49 ILFATITQP 0.010 2228 50 LFATITQPS 0.010 2229 53 TITQPSLHE 0.010 2230 17 LLTGVPGLE 0.010 2231 24 LEAFHTWIS 0.010 2232 60 HEPMYYFLS 0.010 2233 34 PFCFLSVTA 0.001 2234 15 IFLLTGVPG 0.001 2235 26 AFHTWISIP 0.001 2236 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000 0 0 0.000

TABLE XVII V2-B35-9mers: 238P1B2 SEQ Pos 123456789 Score ID 9 TSPLMNPVI 2.000 2237 4 NTYLLTSPL 1.000 2238 2 IANTYLLTS 0.300 2239 8 LTSPLMNPV 0.200 2240 5 TYLLTSPLM 0.200 2241 6 YLLTSPLMN 0.100 2242 1 MIANTYLLT 0.100 2243 7 LLTSPLMNP 0.010 2244 3 ANTYLLTSP 0.010 2245

TABLE XVIII V1-B35-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 99 TPMVALLIRL 20.000 2246 225 ISPLMNPVIY 10.000 2247 118 HSYCYHPDVM 10.000 2248 184 VSHIVAFAIY 10.000 2249 34 ISFNACLSHM 10.000 2250 171 ASPEERKETF 10.000 2251 88 VASVIRGLLM 6.000 2252 146 FSTVGVDLLL 5.000 2253 89 ASVIRGLLML 5.000 2254 53 ESSVLLAMAF 5.000 2255 137 NSAVGLTAMF 5.000 2256 40 LSHMFFIKFF 5.000 2257 15 LSISTLVTML 5.000 2258 208 KQAPAYVHTM 4.000 2259 188 VAFAIYYIPL 3.000 2260 240 QIRRAVIKIL 3.000 2261 133 DTRINSAVGL 3.000 2262 237 KTKQIRRAVI 2.400 2263 14 GLSISTLVTM 2.000 2264 67 AVSNPLRYAM 2.000 2265 210 APAYVHTMIA 2.000 2266 107 RLSYCHSQVL 2.000 2267 213 YVHTMIANTY 2.000 2268 17 ISTLVTMLSI 2.000 2269 226 SPLMNPVIYS 2.000 2270 113 SQVLHHSYCY 2.000 2271 149 VGVDLLLILL 2.000 2272 70 NPLRYAMILT 2.000 2273 220 NTYLLISPLM 2.000 2274 8 LSATDLGLSI 2.000 2275 65 FVAVSNPLRY 2.000 2276 136 INSAVGLTAM 2.000 2277 68 VSNPLRYAMI 2.000 2278 101 MVALLIRLSY 2.000 2279 120 YCYHPDVMKL 1.500 2280 80 DSRIAQIGVA 1.500 2281 74 YANILTDSRI 1.200 2282 172 SPEERKETFS 1.200 2283 209 QAPAYVHTMI 1.200 2284 37 NACLSHMFFI 1.200 2285 83 IAQIGVASVI 1.200 2286 95 LLMLTPMVAL 1.000 2287 20 LVTMLSIFWF 1.000 2288 153 LLLILLSYVL 1.000 2289 191 AIYYIPLISL 1.000 2290 144 AMFSTVGVDL 1.000 2291 215 HTMIANTYLL 1.000 2292 56 VLLAMAFDRF 1.000 2293 49 FTVMESSVLL 1.000 2294 4 FLSMLSATDL 1.000 2295 18 STLVTMLSIF 1.000 2296 219 ANTYLLISPL 1.000 2297 87 GVASVIRGLL 1.000 2298 148 TVGVDLLLIL 1.000 2299 36 FNACLSHMFF 1.000 2300 96 LMLTPMVALL 1.000 2301 158 LSYVLIIRTV 1.000 2302 6 SMLSATDLGL 1.000 2303 69 SNPLRYAMIL 1.000 2304 86 IGVASVIRGL 1.000 2305 39 CLSHMFFIKF 1.000 2306 197 LISLSIVHRF 1.000 2307 181 STCVSHIVAF 1.000 2308 77 ILTDSRIAQI 0.800 2309 239 KQIRRAVIKI 0.800 2310 123 HPDVMKLSCT 0.600 2311 9 SATDLGLSIS 0.600 2312 60 MAFDRFVAVS 0.600 2313 51 VMESSVLLAM 0.600 2314 147 STVGVDLLLI 0.600 2315 30 NVREISFNAC 0.600 2316 129 LSCTDTRINS 0.500 2317 180 FSTCVSHIVA 0.500 2318 112 HSQVLHHSYC 0.500 2319 19 TLVTMLSIFW 0.500 2320 58 LAMAFDRFVA 0.450 2321 170 VASPEERKET 0.450 2322 186 HIVAFAIYYI 0.400 2323 178 ETFSTCVSHI 0.400 2324 216 TMIANTYLLI 0.400 2325 93 RGLLMLTPMV 0.400 2326 25 SIFWFNVREI 0.400 2327 154 LLILLSYVLI 0.400 2328 97 MLTPMVALLI 0.400 2329 155 LILLSYVLII 0.400 2330 82 RIAQIGVASV 0.400 2331 232 VIYSVKTKQI 0.400 2332 183 CVSHIVAFAI 0.400 2333 224 LISPLMNPVI 0.400 2334 190 FAIYYIPLIS 0.300 2335 235 SVKTKQIRRA 0.300 2336 128 KLSCTDTRIN 0.300 2337 66 VAVSNPLRYA 0.300 2338 45 FTKFPTVMES 0.300 2339 102 VALLIRLSYC 0.300 2340 163 IIRTVLSVAS 0.300 2341 245 VIKILHSKET 0.300 2342 138 SAVGLTAMFS 0.300 2343 142 LTAMFSTVGV 0.200 2344 230 NPVIYSVKTK 0.200 2345

TABLE XVIII V1B-B35-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 56 QPSLREPMYY 60.000 2346 61 EPMYYFLSML 20.000 2347 21 VPGLEAFHTW 10.000 2348 41 TALLGNSLIL 3.000 2349 58 SLHEPMYYFL 2.000 2350 33 IPFCFLSVTA 2.000 2351 54 ITQPSLHEPM 2.000 2352 55 TQPSLHEPMY 2.000 2353 39 SVTALLGNSL 1.000 2354 8 NITSTSIIFL 1.000 2355 28 HTWISIPFCF 1.000 2356 31 ISIPFCFLSV 1.000 2357 35 FCFLSVTALL 1.000 2358 7 QNITSTSIIF 1.000 2359 12 TSIIFLLTGV 1.000 2360 42 ALLGNSLILF 1.000 2361 9 ITSTSIIFLL 1.000 2362 18 LTGVPGLEAF 1.000 2363 57 PSLHEPMYYF 0.500 2364 3 TSTLQNITST 0.500 2365 10 TSTSIIFLLT 0.500 2366 38 LSVTALLGNS 0.500 2367 46 NSLILFATIT 0.500 2368 6 LQNITSTSII 0.400 2369 40 VTALLGNSLI 0.400 2370 45 GNSLTLFATI 0.400 2371 5 TLQNITSTSI 0.400 2372 60 HEPMYYFLSM 0.200 2373 20 GVPGLEAFHT 0.150 2374 15 IFLLTGVPGL 0.100 2375 44 LGNSLILFAT 0.100 2376 1 FITSTLQNIT 0.100 2377 32 SIPFCFLSVT 0.100 2378 37 FLSVTALLGN 0.100 2379 30 WISIPFCFLS 0.100 2380 49 ILFATITQPS 0.100 2381 4 STLQNITSTS 0.100 2382 17 LLTGVPGLEA 0.100 2383 50 LFATITQPSL 0.100 2384 43 LLGNSLILFA 0.100 2385 29 TWISIPFCFL 0.100 2386 26 AFHTWISIPF 0.100 2387 2 ITSTLQNITS 0.100 2388 22 PGLEAFHTWI 0.080 2389 24 LEAFHTWISI 0.040 2390 51 FATITQPSLH 0.030 2391 25 EAFHTWISIP 0.030 2392 23 GLEAFHTWIS 0.030 2393 34 PFCFLSVTAL 0.010 2394 27 FHTWISIPFC 0.010 2395 19 TGVPGLEAFH 0.010 2396 16 FLLTGVPGLE 0.010 2397 48 LILFATITQP 0.010 2398 52 ATITQPSLHE 0.010 2399 62 PMYYFLSMLS 0.010 2400 11 STSIIFLLTG 0.010 2401 14 IIFLLTGVPG 0.010 2402 47 SLILFATITQ 0.010 2403 13 SIIFLLTGVP 0.010 2404 53 TITQPSLHEP 0.010 2405 59 LHEPMYYFLS 0.003 2406 36 CFLSVTALLG 0.001 2407 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000 0 0.000

TABLE XVIII V2-B35-10mers: 238P1B2 SEQ Pos 1234567890 Score ID 10 TSPLMNPVIY 10.000 2408 5 NTYLLTSPLM 2.000 2409 4 ANTYLLTSPL 1.000 2410 9 LTSPLMNPVI 0.400 2411 8 LLTSPLMNPV 0.200 2412 1 TMIANTYLLT 0.100 2413 2 MIANTYLLTS 0.100 2414 3 IANTYLLTSP 0.030 2415 7 YLLTSPLMNP 0.010 2416 6 TYLLTSPLMN 0.010 2417

TABLE XIX 238P1B2: HLA Peptide Scoring Results and Position Determination Key Variant 1A: for Nonamers, decamers, and 15-mers, search peptide used (SEQ ID: 2418): MYYFLSMLSA TDLGLSISTL VTMLSIFWFN VREISFNACL SHMFFIKFFT VMESSVLLAM AFDRFVAVSN PLRYAMILTD SRIAQIGVAS VIRGLLMLTP MVALLIRLSY CHSQVLHHSY CYHPDVMKLS CTDTRINSAV GLTAMFSTVG VDLLLILLSY VLIIRTVLSV ASPEERKETF STCVSHIVAF AIYYIPLISL SIVHRFGPQA PAYVHTMIAN TYLLISPLMN PVIYSVKTKQ IRRAVIKILH SKET Variant 1B: search peptides used: Nonamers (aa 1-70) (SEQ ID: 2419) FITSTLQNIT STSIIFLLTG VPGLEAFHTW ISIPFCFLSV TALLGNSLIL FATITQPSLH EPMYYFLSML Decamers (aa 1-71) (SEQ ID: 2420) FITSTLQNIT STSIIFLLTG VPGLEAFHTW ISIPFCFLSV TALLGNSLIL FATITQPSLH EPMYYFLSML S 15-mers (aa 1-76) (SEQ ID: 2421) FITSTLQNIT STSIIFLLTG VPGLEAFHTW ISIPFCFLSV TALLGNSLIL FATITQPSLH EPMYYFLSML SATDLG Variant 2: search peptides used: nonamers (aa 217-233) (SEQ ID: 2422) MIANTYLLTSPLMNPVI decamers (aa 216-234) (SEQ ID: 2423) TMIANTYLLTSPLMNPVIY 15-mers for MHC II (aa 211-239) (SEQ ID: 2424) PAYVHTMIANTYLLTSPLMNPVIYSVKTK Note that variant 1A is referred to as “v.1” in the Tables below; variant 1B is referred to as “v.1B” below; variant 2 is referred to as “v.2”.

TABLE XIXA, PART 1 Pos 1 2 3 4 5 6 7 8 9 score SEQ ID HLA-A*0201 9-mers v.1: 238P1B2 228 L M N P V I Y S V 30 2425 153 L L L I L L S Y V 29 2426 162 L I I R T V L S V 29 2427 156 I L L S Y V L I I 28 2428 94 G L L M L T P M V 27 2429 97 M L T P M V A L L 27 2430 83 I A Q I G V A S V 26 2431 96 L M L T P M V A L 26 2432 154 L L I L L S Y V L 26 2433 12 D L G L S I S T L 25 2434 16 S I S T L V T M L 25 2435 90 S V I R G L L M L 25 2436 224 L I S P L M N P V 24 2437 216 T M I A N T Y L L 23 2438 7 M L S A T D L G L 22 2439 87 G V A S V I R G L 22 2440 141 G L T A M F S T V 22 2441 155 L I L L S Y V L I 22 2442 240 Q I R R A V I K I 22 2443 23 M L S I F W F N V 21 2444 194 Y I P L I S L S I 21 2445 75 A M I L T D S R I 20 2446 78 L T D S R I A Q I 20 2447 91 V I R G L L M L T 20 2448 100 P M V A L L I R L 20 2449 150 G V D L L L I L L 20 2450 187 I V A F A I Y Y I 20 2451 190 F A I Y Y I P L I 20 2452 192 I Y Y I P L I S L 20 2453 220 N T Y L L I S P L 20 2454 18 S T L V T M L S I 19 2455 50 T V M E S S V L L 19 2456 60 M A F D R F V A V 19 2457 107 R L S Y C H S Q V 19 2458 128 K L S C T D T R I 19 2459 143 T A M F S T V G V 19 2460 147 S T V G V D L L L 19 2461 217 M I A N T Y L L I 19 2462 223 L L I S P L M N P 19 2463 9 S A T D L G L S I 18 2464 14 G L S I S T L V T 18 2465 58 L A M A F D R F V 18 2466 103 A L L I R L S Y C 18 2467 121 C Y H P D V M K L 18 2468 149 V G V D L L L I L 18 2469 152 D L L L I L L S Y 18 2470 159 S Y V L I I R T V 18 2471 160 Y V L I I R T V L 18 2472 163 I I R T V L S V A 18 2473 195 I P L I S L S I V 18 2474 51 V M E S S V L L A 17 2475 95 L L M L T P M V A 17 2476 135 R I N S A V G L T 17 2477 148 T V G V D L L L I 17 2478 161 V L I I R T V L S 17 2479 4 F L S M L S A T D 16 2480 15 L S I S T L V T M 16 2481 26 I F W F N V R E I 16 2482 104 L L I R L S Y C H 16 2483 146 F S T V G V D L L 16 2484 197 L I S L S I V H R 16 2485 209 Q A P A Y V H T M 16 2486 213 Y V H T M I A N T 16 2487 32 R E I S F N A C L 15 2488 49 F T V M E S S V L 15 2489 59 A M A F D R F V A 15 2490 67 A V S N P L R Y A 15 2491 88 V A S V I R G L L 15 2492 98 L T P M V A L L I 15 2493 131 C T D T R I N S A 15 2494 134 T R I N S A V G L 15 2495 144 A M F S T V G V D 15 2496 157 L L S Y V L I I R 15 2497 189 A F A I Y Y I P L 15 2498 215 H T M I A N T Y L 15 2499 222 Y L L I S P L M N 15 2500 237 K T K Q I R R A V 15 2501 241 I R R A V I K I L 15 2502 244 A V I K I L H S K 15 2503 6 S M L S A T D L G 14 2504 13 L G L S I S T L V 14 2505 19 T L V T M L S I F 14 2506 25 S I F W F N V R E 14 2507 43 M F F I K F F T V 14 2508 57 L L A M A F D R F 14 2509 76 M I L T D S R I A 14 2510 84 A Q I G V A S V I 14 2511 167 V L S V A S P E E 14 2512 183 C V S H I V A F A 14 2513 3 Y F L S M L S A T 13 2514 5 L S M L S A T D L 13 2515 56 V L L A M A F D R 13 2516 70 N P L R Y A M I L 13 2517 77 I L T D S R I A Q 13 2518 82 R I A Q I G V A S 13 2519 108 L S Y C H S Q V L 13 2520 118 H S Y C Y H P D V 13 2521 137 N S A V G L T A M 13 2522 138 S A V G L T A M F 13 2523 218 I A N T Y L L I S 13 2524 227 P L M N P V I Y S 13 2525 10 A T D L G L S I S 12 2526 38 A C L S H M F F I 12 2527 42 H M F F I K F F T 12 2528 45 F I K F F T V M E 12 2529 48 F F T V M E S S V 12 2530 69 S N P L R Y A M I 12 2531 74 Y A M I L T D S R 12 2532 81 S R I A Q I G V A 12 2533 126 V M K L S C T D T 12 2534 145 M F S T V G V D L 12 2535 158 L S Y V L I I R T 12 2536 165 R T V L S V A S P 12 2537 179 T F S T C V S H I 12 2538 199 S L S I V H R F G 12 2539 201 S I V H R F G K Q 12 2540 206 F G K Q A P A Y V 12 2541 232 V I Y S V K T K Q 12 2542 233 I Y S V K T K Q I 12 2543 11 T D L G L S I S T 11 2544 35 S F N A C L S H M 11 2545 64 R F V A V S N P L 11 2546 68 V S N P L R Y A M 11 2547 101 M V A L L I R L S 11 2548 115 V L H H S Y C Y H 11 2549 180 F S T C V S H I V 11 2550 191 A I Y Y I P L I S 11 2551 193 Y Y I P L I S L S 11 2552 208 K Q A P A Y V H T 11 2553 210 A P A Y V H T M I 11 2554 225 I S P L M N P V I 11 2555 245 V I K I L H S K E 11 2556 2 Y Y F L S M L S A 10 2557 21 V T M L S I F W F 10 2558 22 T M L S I F W F N 10 2559 30 N V R E I S F N A 10 2560 39 C L S H M F F I K 10 2561 52 M E S S V L L A M 10 2562 55 S V L L A M A F D 10 2563 71 P L R Y A M I L T 10 2564 72 L R Y A M I L T D 10 2565 80 D S R I A Q I G V 10 2566 85 Q I G V A S V I R 10 2567 93 R G L L M L T P M 10 2568 102 V A L L I R L S Y 10 2569 105 L I R L S Y C H S 10 2570 132 T D T R I N S A V 10 2571 176 R K E T F S T C V 10 2572 181 S T C V S H I V A 10 2573 184 V S H I V A F A I 10 2574 196 P L I S L S I V H 10 2575 202 I V H R F G K Q A 10 2576 204 H R F G K Q A P A 10 2577 238 T K Q I R R A V I 10 2578 243 R A V I K I L H S 10 2579 61 A F D R F V A V S 9 2580 136 I N S A V G L T A 9 2581 140 V G L T A M F S T 9 2582 142 L T A M F S T V G 9 2583 151 V D L L L I L L S 9 2584 169 S V A S P E E R K 9 2585 170 V A S P E E R K E 9 2586 171 A S P E E R K E T 9 2587 186 H I V A F A I Y Y 9 2588 198 I S L S I V H R F 9 2589 230 N P V I Y S V K T 9 2590 246 I K I L H S K E T 9 2591 8 L S A T D L G L S 8 2592 44 F F I K F F T V M 8 2593 46 I K F F T V M E S 8 2594 53 E S S V L L A M A 8 2595 65 F V A V S N P L R 8 2596 106 I R L S Y C H S Q 8 2597 211 P A Y V H T M I A 8 2598 236 V K T K Q I R R A 8 2599 89 A S V I R G L L M 7 2600 110 Y C H S Q V L H H 7 2601 114 Q V L H H S Y C Y 7 2602 125 D V M K L S C T D 7 2603 182 T C V S H I V A F 7 2604 188 V A F A I Y Y I P 7 2605 219 A N T Y L L I S P 7 2606 221 T Y L L I S P L M 7 2607 231 P V I Y S V K T K 7 2608 20 L V T M L S I F W 6 2609 34 I S F N A C L S H 6 2610 41 S H M F F I K F F 6 2611 66 V A V S N P L R Y 6 2612 73 R Y A M I L T D S 6 2613 86 I Q V A S V I R G 6 2614 92 I R G L L M L T P 6 2615 99 T P M V A L L I R 6 2616 120 Y C Y H P D V M K 6 2617 124 P D V M K L S C T 6 2618 127 M K L S C T D T R 6 2619 139 A V G L T A M F S 6 2620 166 T V L S V A S P E 6 2621 178 E T F S T C V S H 6 2622 185 S H I V A F A I Y 6 2623 226 S P L M N P V I Y 6 2624 235 S V K T K Q I R R 6 2625 29 F N V R E I S F N 5 2626 36 F N A C L S H M F 5 2627 40 L S H M F F I K F 5 2628 111 C H S Q V L H H S 5 2629 119 S Y C Y H P D V M 5 2630 133 D T R I N S A V G 5 2631 164 I R T V L S V A S 5 2632 172 S P E E R K E T F 5 2633 174 E E R K E T F S T 5 2634 200 L S I V H R F G K 5 2635 239 K Q I R R A V I K 5 2636 24 L S I F W F N V R 4 2637 33 E I S F N A C L S 4 2638 37 N A C L S H M F F 4 2639 47 K F F T V M E S S 4 2640 54 S S V L L A M A F 4 2641 62 F D R F V A V S N 4 2642 63 D R F V A V S N P 4 2643 109 S Y C H S Q V L H 4 2644 112 H S Q V L H H S Y 4 2645 116 L H H S Y C Y H P 4 2646 129 L S C T D T R I N 4 2647 168 L S V A S P E E R 4 2648 17 I S T L V T M L S 3 2649 28 W F N V R E I S F 3 2650 123 H P D V M K L S C 3 2651 203 V H R F G K Q A P 3 2652 207 G K Q A P A Y V H 3 2653 212 A Y V H T M I A N 3 2654 214 V H T M I A N T Y 3 2655 27 F W F N V R E I S 2 2656 31 V R E I S F N A C 2 2657 113 S Q V L H H S Y C 2 2658 122 Y H P D V M K L S 2 2659 130 S C T D T R I N S 2 2660 205 R F G K Q A P A Y 2 2661 234 Y S V K T K Q I R 2 2662 242 R R A V I K I L H 2 2663 1 M Y Y F L S M L S 1 2664 177 K E T F S T C V S 1 2665 173 P E E R K E T F S −3 2666 HLA-A1 9-mers v.1: 238P1B2 102 V A L L I R L S Y 23 2667 66 V A V S N P L R Y 22 2668 152 D L L L I L L S Y 22 2669 51 V M E S S V L L A 20 2670 185 S H I V A F A I Y 20 2671 10 A T D L G L S I S 19 2672 112 H S Q V L H H S Y 19 2673 147 S T V G V D L L L 19 2674 186 H I V A F A I Y Y 19 2675 205 R F G K Q A P A Y 17 2676 226 S P L M N P V I Y 17 2677 78 L T D S R I A Q I 16 2678 98 L T P M V A L L I 16 2679 123 H P D V M K L S C 16 2680 131 C T D T R I N S A 16 2681 150 G V D L L L I L L 16 2682 214 V H T M I A N T Y 16 2683 114 Q V L H H S Y C Y 15 2684 18 S T L V T M L S I 14 2685 31 V R E I S F N A C 14 2686 89 A S V I R G L L M 14 2687 40 L S H M F F I K F 13 2688 61 A F D R F V A V S 13 2689 181 S T C V S H I V A 13 2690 172 S P E E R K E T F 12 2691 90 S V I R G L L M L 11 2692 110 Y C H S Q V L H H 11 2693 34 I S F N A C L S H 10 2694 68 V S N P L R Y A M 10 2695 146 F S T V G V D L L 10 2696 173 P E E R K E T F S 10 2697 176 R K E T F S T C V 10 2698 222 Y L L I S P L M N 10 2699 9 S A T D L G L S I 9 2700 136 I N S A V G L T A 9 2701 151 V D L L L I L L S 9 2702 156 I L L S Y V L I I 9 2703 2 Y Y F L S M L S A 8 2704 14 G L S I S T L V T 8 2705 17 I S T L V T M L S 8 2706 24 L S I F W F N V R 8 2707 49 F T V M E S S V L 8 2708 52 M E S S V L L A M 8 2709 80 D S R I A Q I G V 8 2710 97 M L T P M V A L L 8 2711 148 T V G V D L L L I 8 2712 158 L S Y V L I I R T 8 2713 193 Y Y I P L I S L S 8 2714 194 Y I P L I S L S I 8 2715 217 M I A N T Y L L I 8 2716 7 M L S A T D L G L 7 2717 21 V T M L S I F W F 7 2718 71 P L R Y A M I L T 7 2719 121 C Y H P D V M K L 7 2720 130 S C T D T R I N S 7 2721 133 D T R I N S A V G 7 2722 142 L T A M F S T V G 7 2723 149 V G V D L L L I L 7 2724 157 L L S Y V L I I R 7 2725 162 L I I R T V L S V 7 2726 165 R T V L S V A S P 7 2727 170 V A S P E E R K E 7 2728 180 F S T C V S H I V 7 2729 184 V S H I V A F A I 7 2730 191 A I Y Y I P L I S 7 2731 201 S I V H R F G K Q 7 2732 215 H T M I A N T Y L 7 2733 218 I A N T Y L L I S 7 2734 227 P L M N P V I Y S 7 2735 237 K T K Q I R R A V 7 2736 240 Q I R R A V I K I 7 2737 8 L S A T D L G L S 6 2738 54 S S V L L A M A F 6 2739 72 L R Y A M I L T D 6 2740 77 I L T D S R I A Q 6 2741 92 I R G L L M L T P 6 2742 99 T P M V A L L I R 6 2743 122 Y H P D V M K L S 6 2744 137 N S A V G L T A M 6 2745 171 A S P E E R K E T 6 2746 178 E T F S T C V S H 6 2747 189 A F A I Y Y I P L 6 2748 196 P L I S L S I V H 6 2749 220 N T Y L L I S P L 6 2750 225 I S P L M N P V I 6 2751 228 L M N P V I Y S V 6 2752 234 Y S V K T K Q I R 6 2753 235 S V K T K Q I R R 6 2754 242 R R A V I K I L H 6 2755 243 R A V I K I L H S 6 2756 11 T D L G L S I S T 5 2757 44 F F I K F F T V M 5 2758 53 E S S V L L A M A 5 2759 81 S R I A Q I G V A 5 2760 84 A Q I G V A S V I 5 2761 86 I G V A S V I R G 5 2762 108 L S Y C H S Q V L 5 2763 118 H S Y C Y H P D V 5 2764 192 I Y Y I P L I S L 5 2765 198 I S L S I V H R F 5 2766 199 S L S I V H R F G 5 2767 200 L S I V H R F G K 5 2768 212 A Y V H T M I A N 5 2769 219 A N T Y L L I S P 5 2770 232 V I Y S V K T K Q 5 2771 5 L S M L S A T D L 4 2772 6 S M L S A T D L G 4 2773 15 L S I S T L V T M 4 2774 16 S I S T L V T M L 4 2775 20 L V T M L S I F W 4 2776 27 F W F N V R E I S 4 2777 28 W F N V R E I S F 4 2778 39 C L S H M F F I K 4 2779 57 L L A M A F D R F 4 2780 65 F V A V S N P L R 4 2781 88 V A S V I R G L L 4 2782 91 V I R G L L M L T 4 2783 100 P M V A L L I R L 4 2784 109 S Y C H S Q V L H 4 2785 120 Y C Y H P D V M K 4 2786 129 L S C T D T R I N 4 2787 138 S A V G L T A M F 4 2788 141 G L T A M F S T V 4 2789 160 Y V L I I R T V L 4 2790 161 V L I I R T V L S 4 2791 168 L S V A S P E E R 4 2792 190 F A I Y Y I P L I 4 2793 208 K Q A P A Y V H T 4 2794 210 A P A Y V H T M I 4 2795 224 L I S P L M N P V 4 2796 22 T M L S I F W F N 3 2797 25 S I F W F N V R E 3 2798 43 M F F I K F F T V 3 2799 59 A M A F D R F V A 3 2800 60 M A F D R F V A V 3 2801 62 F D R F V A V S N 3 2802 69 S N P L R Y A M I 3 2803 83 I A Q I G V A S V 3 2804 119 S Y C Y H P D V M 3 2805 135 R I N S A V G L T 3 2806 144 A M F S T V G V D 3 2807 145 M F S T V G V D L 3 2808 163 I I R T V L S V A 3 2809 169 S V A S P E E R K 3 2810 175 E R K E T F S T C 3 2811 203 V H R F G K Q A P 3 2812 230 N P V I Y S V K T 3 2813 236 V K T K Q I R R A 3 2814 245 V I K I L H S K E 3 2815 1 M Y Y F L S M L S 2 2816 3 Y F L S M L S A T 2 2817 4 F L S M L S A T D 2 2818 13 L G L S I S T L V 2 2819 33 E I S F N A C L S 2 2820 35 S F N A C L S H M 2 2821 36 F N A C L S H M F 2 2822 41 S H M F F I K F F 2 2823 45 F I K F F T V M E 2 2824 47 K F F T V M E S S 2 2825 50 T V M E S S V L L 2 2826 55 S V L L A M A F D 2 2827 56 V L L A M A F D R 2 2828 64 R F V A V S N P L 2 2829 67 A V S N P L R Y A 2 2830 74 Y A M I L T D S R 2 2831 87 G V A S V I R G L 2 2832 94 G L L M L T P M V 2 2833 95 L L M L T P M V A 2 2834 101 M V A L L I R L S 2 2835 103 A L L I R L S Y C 2 2836 111 C H S Q V L H H S 2 2837 113 S Q V L H H S Y C 2 2838 115 V L H H S Y C Y H 2 2839 154 L L I L L S Y V L 2 2840 155 L I L L S Y V L I 2 2841 159 S Y V L I I R T V 2 2842 166 T V L S V A S P E 2 2843 167 V L S V A S P E E 2 2844 207 G K Q A P A Y V H 2 2845 209 Q A P A Y V H T M 2 2846 213 Y V H T M I A N T 2 2847 216 T M I A N T Y L L 2 2848 221 T Y L L I S P L M 2 2849 223 L L I S P L M N P 2 2850 238 T K Q I R R A V I 2 2851 241 I R R A V I K I L 2 2852 12 D L G L S I S T L 1 2853 19 T L V T M L S I F 1 2854 23 M L S I F W F N V 1 2855 26 I F W F N V R E I 1 2856 29 F N V R E I S F N 1 2857 32 R E I S F N A C L 1 2858 38 A C L S H M F F I 1 2859 46 I K F F T V M E S 1 2860 48 P F T V M E S S V 1 2861 63 D R F V A V S N P 1 2862 70 N P L R Y A M I L 1 2863 75 A M I L T D S R I 1 2864 82 R I A Q I G V A S 1 2865 96 L M L T P M V A L 1 2866 104 L L I R L S Y C H 1 2867 106 I R L S Y C H S Q 1 2868 107 R L S Y C H S Q V 1 2869 116 L H H S Y C Y H P 1 2870 126 V M K L S C T D T 1 2871 128 K L S C T D T R I 1 2872 132 T D T R I N S A V 1 2873 139 A V G L T A M F S 1 2874 140 V G L T A M F S T 1 2875 153 L L L I L L S Y V 1 2876 177 K E T F S T C V S 1 2877 179 T F S T C V S H I 1 2878 183 C V S H I V A F A 1 2879 188 V A F A I Y Y I P 1 2880 202 I V H R F G K Q A 1 2881 204 H R F G K Q A P A 1 2882 206 F G K Q A P A Y V 1 2883 229 M N P V I Y S V K 1 2884 231 P V I Y S V K T K 1 2885 244 A V I K I L H S K 1 2886 HLA-A26 9-mers v.1: 238P1B2 152 D L L L I L L S Y 29 2887 12 D L G L S I S T L 28 2888 21 V T M L S I F W F 25 2889 87 G V A S V I R G L 25 2890 90 S V I R G L L M L 25 2891 16 S I S T L V T M L 24 2892 57 L L A M A F D R F 24 2893 150 G V D L L L I L L 24 2894 220 N T Y L L I S P L 24 2895 19 T L V T M L S I F 23 2896 97 M L T P M V A L L 23 2897 50 T V M E S S V L L 22 2898 44 F F I K F F T V M 21 2899 178 E T F S T C V S H 21 2900 35 S F N A C L S H M 20 2901 114 Q V L H H S Y C Y 20 2902 147 S T V G V D L L L 20 2903 186 H I V A F A I Y Y 20 2904 205 R F G K Q A P A Y 20 2905 49 F T V M E S S V L 19 2906 154 L L I L L S Y V L 19 2907 189 A F A I Y Y I P L 19 2908 78 L T D S R I A Q I 18 2909 91 V I R G L L M L T 18 2910 125 D V M K L S C T D 18 2911 160 Y V L I I R T V L 18 2912 215 H T M I A N T Y L 18 2913 244 A V I K I L H S K 18 2914 7 M L S A T D L G L 17 2915 28 W F N V R E I S F 17 2916 133 D T R I N S A V G 17 2917 162 L I I R T V L S V 17 2918 165 R T V L S V A S P 17 2919 182 T C V S H I V A F 17 2920 185 S H I V A F A I Y 17 2921 197 L I S L S I V H R 17 2922 223 L L I S P L M N P 17 2923 224 L I S P L M N P V 17 2924 231 P V I Y S V K T K 17 2925 10 A T D L G L S I S 16 2926 15 L S I S T L V T M 16 2927 33 E I S F N A C L S 16 2928 101 M V A L L I R L S 16 2929 105 L I R L S Y C H S 16 2930 121 C Y H P D V M K L 16 2931 131 C T D T R I N S A 16 2932 155 L I L L S Y V L I 16 2933 198 I S L S I V H R F 16 2934 213 Y V H T M I A N T 16 2935 18 S T L V T M L S I 15 2936 40 L S H M F F I K F 15 2937 55 S V L L A M A F D 15 2938 135 R I N S A V G L T 15 2939 138 S A V G L T A M F 15 2940 145 M F S T V G V D L 15 2941 163 I I R T V L S V A 15 2942 183 C V S H I V A F A 15 2943 187 I V A F A I Y Y I 15 2944 201 S I V H R F G K Q 15 2945 217 M I A N T Y L L I 15 2946 240 Q I R R A V I K I 15 2947 25 S I F W F N V R E 14 2948 41 S H M F F I K F F 14 2949 52 M E S S V L L A M 14 2950 63 D R F V A V S N P 14 2951 64 R F V A V S N P L 14 2952 67 A V S N P L R Y A 14 2953 82 R I A Q I G V A S 14 2954 96 L M L T P M V A L 14 2955 100 P M V A L L I R L 14 2956 137 N S A V G L T A M 14 2957 148 T V G V D L L L I 14 2958 149 V G V D L L L I L 14 2959 153 L L L I L L S Y V 14 2960 175 E R K E T F S T C 14 2961 245 V I K I L H S K E 14 2962 3 Y F L S M L S A T 13 2963 30 N V R E I S F N A 13 2964 36 F N A C L S H M F 13 2965 43 M F F I K F F T V 13 2966 45 F I K F F T V M E 13 2967 47 K F F T V M E S S 13 2968 53 E S S V L L A M A 13 2969 54 S S V L L A M A F 13 2970 65 F V A V S N P L R 13 2971 103 A L L I R L S Y C 13 2972 115 V L H H S Y C Y H 13 2973 134 T R I N S A V G L 13 2974 141 G L T A M F S T V 13 2975 156 I L L S Y V L I I 13 2976 169 S V A S P E E R K 13 2977 192 I Y Y I P L I S L 13 2978 209 Q A P A Y V H T M 13 2979 235 S V K T K Q I R R 13 2980 237 K T K Q I R R A V 13 2981 32 R E I S F N A C L 12 2982 37 N A C L S H M F F 12 2983 39 C L S H M F F I K 12 2984 61 A F D R F V A V S 12 2985 66 V A V S N P L R Y 12 2986 68 V S N P L R Y A M 12 2987 71 P L R Y A M I L T 12 2988 93 R G L L M L T P M 12 2989 112 H S Q V L H H S Y 12 2990 146 F S T V G V D L L 12 2991 157 L L S Y V L I I R 12 2992 166 T V L S V A S P E 12 2993 172 S P E E R K E T F 12 2994 179 T F S T C V S H I 12 2995 194 Y I P L I S L S I 12 2996 196 P L I S L S I V H 12 2997 227 P L M N P V I Y S 12 2998 232 V I Y S V K T K Q 12 2999 241 I R R A V I K I L 12 3000 20 L V T M L S I F W 11 3001 26 I F W F N V R E I 11 3002 77 I L T D S R I A Q 11 3003 85 Q I G V A S V I R 11 3004 94 G L L M L T P M V 11 3005 98 L T P M V A L L I 11 3006 102 V A L L I R L S Y 11 3007 104 L L I R L S Y C H 11 3008 139 A V G L T A M F S 11 3009 142 L T A M F S T V G 11 3010 161 V L I I R T V L S 11 3011 181 S T C V S H I V A 11 3012 202 I V H R F G K Q A 11 3013 226 S P L M N P V I Y 11 3014 4 F L S M L S A T D 10 3015 76 M I L T D S R I A 10 3016 107 R L S Y C H S Q V 10 3017 167 V L S V A S P E E 10 3018 191 A I Y Y I P L I S 10 3019 193 Y Y I P L I S L S 10 3020 214 V H T M I A N T Y 10 3021 216 T M I A N T Y L L 10 3022 222 Y L L I S P L M N 10 3023 5 L S M L S A T D L 9 3024 14 G L S I S T L V T 9 3025 23 M L S I F W F N V 9 3026 60 M A F D R F V A V 9 3027 70 N P L R Y A M I L 9 3028 95 L L M L T P M V A 9 3029 108 L S Y C H S Q V L 9 3030 128 K L S C T D T R I 9 3031 208 K Q A P A Y V H T 9 3032 221 T Y L L I S P L M 9 3033 2 Y Y F L S M L S A 8 3034 46 I K F F T V M E S 8 3035 48 F F T V M E S S V 8 3036 56 V L L A M A F D R 8 3037 88 V A S V I R G L L 8 3038 89 A S V I R G L L M 8 3039 119 S Y C Y H P D V M 8 3040 174 E E R K E T F S T 8 3041 199 S L S I V H R F G 8 3042 69 S N P L R Y A M I 7 3043 80 D S R I A Q I G V 7 3044 81 S R I A Q I G V A 7 3045 110 Y C H S Q V L H H 7 3046 144 A M F S T V G V D 7 3047 190 F A I Y Y I P L I 7 3048 228 L M N P V I Y S V 7 3049 236 V K T K Q I R R A 7 3050 24 L S I F W F N V R 6 3051 31 V R E I S F N A C 6 3052 51 V M E S S V L L A 6 3053 83 I A Q I G V A S V 6 3054 86 I G V A S V I R G 6 3055 111 C H S Q V L H H S 6 3056 122 Y H P D V M K L S 6 3057 124 P D V M K L S C T 6 3058 158 L S Y V L I I R T 6 3059 188 V A F A I Y Y I P 6 3060 195 I P L I S L S I V 6 3061 204 H R F G K Q A P A 6 3062 219 A N T Y L L I S P 6 3063 229 M N P V I Y S V K 6 3064 243 R A V I K I L H S 6 3065 8 L S A T D L G L S 5 3066 22 T M L S I F W F N 5 3067 29 F N V R E I S F N 5 3068 38 A C L S H M F F I 5 3069 73 R Y A M I L T D S 5 3070 84 A Q I G V A S V I 5 3071 92 I R G L L M L T P 5 3072 140 V G L T A M F S T 5 3073 151 V D L L L I L L S 5 3074 212 A Y V H T M I A N 5 3075 11 T D L G L S I S T 4 3076 72 L R Y A M I L T D 4 3077 75 A M I L T D S R I 4 3078 99 T P M V A L L I R 4 3079 117 H H S Y C Y H P D 4 3080 159 S Y V L I I R T V 4 3081 171 A S P E E R K E T 4 3082 218 I A N T Y L L I S 4 3083 9 S A T D L G L S I 3 3084 34 I S F N A C L S H 3 3085 143 T A M F S T V G V 3 3086 164 I R T V L S V A S 3 3087 170 V A S P E E R K E 3 3088 200 L S I V H R F G K 3 3089 206 F G K Q A P A Y V 3 3090 239 K Q I R R A V I K 3 3091 246 I K I L H S K E T 3 3092 1 M Y Y F L S M L S 2 3093 27 F W F N V R E I S 2 3094 42 H M F F I K F F T 2 3095 62 F D R F V A V S N 2 3096 74 Y A M I L T D S R 2 3097 106 I R L S Y C H S Q 2 3098 109 S Y G H S Q V L H 2 3099 116 L H H S Y C Y H P 2 3100 120 Y C Y H P D V M K 2 3101 123 H P D V M K L S C 2 3102 126 V M K L S C T D T 2 3103 130 S C T D T R I N S 2 3104 132 T D T R I N S A V 2 3105 168 L S V A S P E E R 2 3106 203 V H R F G K Q A P 2 3107 210 A P A Y V H T M I 2 3108 225 I S P L M N P V I 2 3109 230 N P V I Y S V K T 2 3110 233 I Y S V K T K Q I 2 3111 234 Y S V K T K Q I R 2 3112 6 S M L S A T D L G 1 3113 13 L G L S I S T L V 1 3114 17 I S T L V T M L S 1 3115 59 A M A F D R F V A 1 3116 79 T D S R I A Q I G 1 3117 113 S Q V L H H S Y C 1 3118 118 H S Y C Y H P D V 1 3119 127 M K L S C T D T R 1 3120 173 P E E R K E T F S 1 3121 177 K E T F S T C V S 1 3122 184 V S H I V A F A I 1 3123 211 P A Y V H T M I A 1 3124 238 T K Q I R R A V I 1 3125 242 R R A V I K I L H 1 3126 HLA-A3 9-mers v.1: 238P1B2 239 K Q I R R A V I K 28 3127 244 A V I K I L H S K 28 3128 231 P V I Y S V K T K 27 3129 152 D L L L I L L S Y 24 3130 90 S V I R G L L M L 23 3131 169 S V A S P E E R K 22 3132 56 V L L A M A F D R 21 3133 82 R I A Q I G V A S 21 3134 103 A L L I R L S Y C 21 3135 163 I I R T V L S V A 21 3136 196 P L I S L S I V H 21 3137 202 I V H R F G K Q A 21 3138 39 C L S H M F F I K 20 3139 104 L L I R L S Y C H 20 3140 107 R L S Y C H S Q V 20 3141 114 Q V L H H S Y C Y 20 3142 161 V L I I R T V L S 20 3143 84 A Q I G V A S V I 19 3144 120 Y C Y H P D V M K 19 3145 141 G L T A M F S T V 19 3146 160 Y V L I I R T V L 19 3147 162 L I I R T V L S V 19 3148 166 T V L S V A S P E 19 3149 186 H I V A F A I Y Y 19 3150 191 A I Y Y I P L I S 19 3151 12 D L G L S I S T L 18 3152 14 G L S I S T L V T 18 3153 85 Q I G V A S V I R 18 3154 139 A V G L T A M F S 18 3155 154 L L I L L S Y V L 18 3156 222 Y L L I S P L M N 18 3157 4 F L S M L S A T D 17 3158 19 T L V T M L S I F 17 3159 55 S V L L A M A F D 17 3160 95 L L M L T P M V A 17 3161 156 I L L S Y V L I I 17 3162 197 L I S L S I V H R 17 3163 229 M N P V I Y S V K 17 3164 235 S V K T K Q I R R 17 3165 97 M L T P M V A L L 16 3166 153 L L L I L L S Y V 16 3167 240 Q I R R A V I K I 16 3168 7 M L S A T D L G L 15 3169 50 T V M E S S V L L 15 3170 57 L L A M A F D R F 15 3171 65 F V A V S N P L R 15 3172 77 I L T D S R I A Q 15 3173 102 V A L L I R L S Y 15 3174 136 I N S A V G L T A 15 3175 155 L I L L S Y V L I 15 3176 157 L L S Y V L I I R 15 3177 30 N V R E I S F N A 14 3178 45 F I K F F T V M E 14 3179 67 A V S N P L R Y A 14 3180 91 V I R G L L M L T 14 3181 115 V L H H S Y C Y H 14 3182 125 D V M K L S C T D 14 3183 128 K L S C T D T R I 14 3184 194 Y I P L I S L S I 14 3185 200 L S I V H R F G K 14 3186 205 R F G K Q A P A Y 14 3187 223 L L I S P L M N P 14 3188 226 S P L M N P V I Y 14 3189 232 V I Y S V K T K Q 14 3190 25 S I F W F N V R E 13 3191 61 A F D R F V A V S 13 3192 62 F D R F V A V S N 13 3193 71 P L R Y A M I L T 13 3194 72 L R Y A M I L T D 13 3195 94 G L L M L T P M V 13 3196 135 R I N S A V G L T 13 3197 148 T V G V D L L L I 13 3198 183 C V S H I V A F A 13 3199 185 S H I V A F A I Y 13 3200 187 I V A F A I Y Y I 13 3201 199 S L S I V H R F G 13 3202 207 G K Q A P A Y V H 13 3203 213 Y V H T M I A N T 13 3204 214 V H T M I A N T Y 13 3205 217 M I A N T Y L L I 13 3206 245 V I K I L H S K E 13 3207 34 I S F N A C L S H 12 3208 87 G V A S V I R G L 12 3209 89 A S V I R G L L M 12 3210 101 M V A L L I R L S 12 3211 105 L I R L S Y C H S 12 3212 172 S P E E R K E T F 12 3213 201 S I V H R F G K Q 12 3214 15 L S I S T L V T M 11 3215 16 S I S T L V T M L 11 3216 23 M L S I F W F N V 11 3217 24 L S I F W F N V R 11 3218 33 E I S F N A C L S 11 3219 54 S S V L L A M A F 11 3220 81 S R I A Q I G V A 11 3221 133 D T R I N S A V G 11 3222 134 T R I N S A V G L 11 3223 138 S A V G L T A M F 11 3224 150 G V D L L L I L L 11 3225 208 K Q A P A Y V H T 11 3226 224 L I S P L M N P V 11 3227 227 P L M N P V I Y S 11 3228 238 T K Q I R R A V I 11 3229 9 S A T D L G L S I 10 3230 20 L V T M L S I F W 10 3231 32 R E I S F N A C L 10 3232 66 V A V S N P L R Y 10 3233 75 A M I L T D S R I 10 3234 76 M I L T D S R I A 10 3235 83 I A Q I G V A S V 10 3236 92 I R G L L M L T P 10 3237 127 M K L S C T D T R 10 3238 165 R T V L S V A S P 10 3239 167 V L S V A S P E E 10 3240 178 E T F S T C V S H 10 3241 10 A T D L G L S I S 9 3242 59 A M A F D R F V A 9 3243 70 N P L R Y A M I L 9 3244 109 S Y C H S Q V L H 9 3245 175 E R K E T F S T C 9 3246 182 T C V S H I V A F 9 3247 192 I Y Y I P L I S L 9 3248 193 Y Y I P L I S L S 9 3249 242 R R A V I K I L H 9 3250 243 R A V I K I L H S 9 3251 18 S T L V T M L S I 8 3252 28 W F N V R E I S F 8 3253 44 F F I K F F T V M 8 3254 60 M A F D R F V A V 8 3255 69 S N P L R Y A M I 8 3256 78 L T D S R I A Q I 8 3257 93 R G L L M L T P M 8 3258 108 L S Y C H S Q V L 8 3259 112 H S Q V L H H S Y 8 3260 144 A M F S T V G V D 8 3261 145 M F S T V G V D L 8 3262 195 I P L I S L S I V 8 3263 198 I S L S I V H R F 8 3264 209 Q A P A Y V H T M 8 3265 220 N T Y L L I S P L 8 3266 241 I R R A V I K I L 8 3267 49 F T V M E S S V L 7 3268 64 R F V A V S N P L 7 3269 74 Y A M I L T D S R 7 3270 96 L M L T P M V A L 7 3271 98 L T P M V A L L I 7 3272 99 T P M V A L L I R 7 3273 110 Y C H S Q V L H H 7 3274 132 T D T R I N S A V 7 3275 147 S T V G V D L L L 7 3276 164 I R T V L S V A S 7 3277 168 L S V A S P E E R 7 3278 177 K E T F S T C V S 7 3279 210 A P A Y V H T M I 7 3280 225 I S P L M N P V I 7 3281 2 Y Y F L S M L S A 6 3282 3 Y F L S M L S A T 6 3283 11 T D L G L S I S T 6 3284 21 V T M L S I F W F 6 3285 29 F N V R E I S F N 6 3286 35 S F N A C L S H M 6 3287 36 F N A C L S H M F 6 3288 37 N A C L S H M F F 6 3289 43 M F F I K F F T V 6 3290 47 K F F T V M E S S 6 3291 73 R Y A M I L T D S 6 3292 79 T D S R I A Q I G 6 3293 106 I R L S Y C H S Q 6 3294 119 S Y C Y H P D V M 6 3295 142 L T A M F S T V G 6 3296 151 V D L L L I L L S 6 3297 159 S Y V L I I R T V 6 3298 184 V S H I V A F A I 6 3299 203 V H R F G K Q A P 6 3300 204 H R F G K Q A P A 6 3301 228 L M N P V I Y S V 6 3302 233 I Y S V K T K Q I 6 3303 237 K T K Q I R R A V 6 3304 246 I K I L H S K E T 6 3305 1 M Y Y F L S M L S 5 3306 5 L S M L S A T D L 5 3307 17 I S T L V T M L S 5 3308 38 A C L S H M F F I 5 3309 41 S H M F F I K F F 5 3310 48 F F T V M E S S V 5 3311 52 M E S S V L L A M 5 3312 68 V S N P L R Y A M 5 3313 113 S Q V L H H S Y C 5 3314 123 H P D V M K L S C 5 3315 137 N S A V G L T A M 5 3316 171 A S P E E R K E T 5 3317 173 P E E R K E T F S 5 3318 174 E E R K E T F S T 5 3319 176 R K E T F S T C V 5 3320 181 S T C V S H I V A 5 3321 189 A F A I Y Y I P L 5 3322 206 F G K Q A P A Y V 5 3323 211 P A Y V H T M I A 5 3324 216 T M I A N T Y L L 5 3325 218 I A N T Y L L I S 5 3326 219 A N T Y L L I S P 5 3327 230 N P V I Y S V K T 5 3328 40 L S H M F F I K F 4 3329 53 E S S V L L A M A 4 3330 80 D S R I A Q I G V 4 3331 86 I G V A S V I R G 4 3332 118 H S Y C Y H P D V 4 3333 121 C Y H P D V M K L 4 3334 124 P D V M K L S C T 4 3335 131 C T D T R I N S A 4 3336 140 V G L T A M F S T 4 3337 143 T A M F S T V G V 4 3338 149 V G V D L L L I L 4 3339 158 L S Y V L I I R T 4 3340 212 A Y V H T M I A N 4 3341 234 Y S V K T K Q I R 4 3342 6 S M L S A T D L G 3 3343 13 L G L S I S T L V 3 3344 27 F W F N V R E I S 3 3345 46 I K F F T V M E S 3 3346 51 V M E S S V L L A 3 3347 63 D R F V A V S N P 3 3348 126 V M K L S C T D T 3 3349 130 S C T D T R I N S 3 3350 170 V A S P E E R K E 3 3351 179 T F S T C V S H I 3 3352 190 F A I Y Y I P L I 3 3353 221 T Y L L I S P L M 3 3354 8 L S A T D L G L S 2 3355 22 T M L S I F W F N 2 3356 26 I F W F N V R E I 2 3357 31 V R E I S F N A C 2 3358 58 L A M A F D R F V 2 3359 88 V A S V I R G L L 2 3360 100 P M V A L L I R L 2 3361 122 Y H P D V M K L S 2 3362 146 F S T V G V D L L 2 3363 188 V A F A I Y Y I P 2 3364 215 H T M I A N T Y L 2 3365 111 C H S Q V L H H S 1 3366 180 F S T C V S H I V 1 3367 HLA-B*0702 9-mers v.1: 238P1B2 70 N P L R Y A M I L 20 3368 210 A P A Y V H T M I 20 3369 230 N P V I Y S V K T 18 3370 195 I P L I S L S I V 17 3371 96 L M L T P M V A L 16 3372 172 S P E E R K E T F 16 3373 7 M L S A T D L G L 15 3374 145 M F S T V G V D L 15 3375 16 S I S T L V T M L 14 3376 147 S T V G V D L L L 14 3377 189 A F A I Y Y I P L 14 3378 192 I Y Y I P L I S L 14 3379 241 I R R A V I K I L 14 3380 50 T V M E S S V L L 13 3381 64 R F V A V S N P L 13 3382 67 A V S N P L R Y A 13 3383 88 V A S V I R G L L 13 3384 97 M L T P M V A L L 13 3385 99 T P M V A L L I R 13 3386 123 H P D V M K L S C 13 3387 136 I N S A V G L T A 13 3388 160 Y V L I I R T V L 13 3389 5 L S M L S A T D L 12 3390 14 G L S I S T L V T 12 3391 32 R E I S F N A C L 12 3392 90 S V I R G L L M L 12 3393 121 C Y H P D V M K L 12 3394 134 T R I N S A V G L 12 3395 149 V G V D L L L I L 12 3396 215 H T M I A N T Y L 12 3397 216 T M I A N T Y L L 12 3398 226 S P L M N P V I Y 12 3399 12 D L G L S I S T L 11 3400 49 F T V M E S S V L 11 3401 52 M E S S V L L A M 11 3402 59 A M A F D R F V A 11 3403 84 A Q I G V A S V I 11 3404 87 G V A S V I R G L 11 3405 89 A S V I R G L L M 11 3406 100 P M V A L L I R L 11 3407 108 L S Y C H S Q V L 11 3408 146 F S T V G V D L L 11 3409 150 G V D L L L I L L 11 3410 154 L L I L L S Y V L 11 3411 174 E E R K E T F S T 11 3412 183 C V S H I V A F A 11 3413 208 K Q A P A Y V H T 11 3414 220 N T Y L L I S P L 11 3415 44 F F I K F F T V M 10 3416 53 E S S V L L A M A 10 3417 60 M A F D R F V A V 10 3418 91 V I R G L L M L T 10 3419 128 K L S C T D T R I 10 3420 138 S A V G L T A M F 10 3421 143 T A M F S T V G V 10 3422 156 I L L S Y V L I I 10 3423 163 I I R T V L S V A 10 3424 204 H R F G K Q A P A 10 3425 224 L I S P L M N P V 10 3426 233 I Y S V K T K Q I 10 3427 9 S A T D L G L S I 9 3428 38 A C L S H M F F I 9 3429 51 V M E S S V L L A 9 3430 58 L A M A F D R F V 9 3431 68 V S N P L R Y A M 9 3432 83 I A Q I G V A S V 9 3433 93 R G L L M L T P M 9 3434 107 R L S Y C H S Q V 9 3435 137 N S A V G L T A M 9 3436 148 T V G V D L L L I 9 3437 162 L I I R T V L S V 9 3438 171 A S P E E R K E T 9 3439 179 T F S T C V S H I 9 3440 182 T C V S H I V A F 9 3441 217 M I A N T Y L L I 9 3442 225 I S P L M N P V I 9 3443 237 K T K Q I R R A V 9 3444 238 T K Q I R R A V I 9 3445 240 Q I R R A V I K I 9 3446 2 Y Y F L S M L S A 8 3447 11 T D L G L S I S T 8 3448 15 L S I S T L V T M 8 3449 18 S T L V T M L S I 8 3450 23 M L S I F W F N V 8 3451 26 I F W F N V R E I 8 3452 30 N V R E I S F N A 8 3453 41 S H M F F I K F F 8 3454 57 L L A M A F D R F 8 3455 71 P L R Y A M I L T 8 3456 75 A M I L T D S R I 8 3457 78 L T D S R I A Q I 8 3458 80 D S R I A Q I G V 8 3459 81 S R I A Q I G V A 8 3460 94 G L L M L T P M V 8 3461 95 L L M L T P M V A 8 3462 98 L T P M V A L L I 8 3463 135 R I N S A V G L T 8 3464 141 G L T A M F S T V 8 3465 155 L I L L S Y V L I 8 3466 176 R K E T F S T C V 8 3467 187 I V A F A I Y Y I 8 3468 194 Y I P L I S L S I 8 3469 198 I S L S I V H R F 8 3470 202 I V H R F G K Q A 8 3471 209 Q A P A Y V H T M 8 3472 3 Y F L S M L S A T 7 3473 13 L G L S I S T L V 7 3474 21 V T M L S I F W F 7 3475 35 S F N A C L S H M 7 3476 36 F N A C L S H M F 7 3477 37 N A C L S H M F F 7 3478 40 L S H M F F I K F 7 3479 42 H M F F I K F F T 7 3480 43 M F F I K F F T V 7 3481 54 S S V L L A M A F 7 3482 69 S N P L R Y A M I 7 3483 118 H S Y C Y H P D V 7 3484 119 S Y C Y H P D V M 7 3485 126 V M K L S C T D T 7 3486 131 C T D T R I N S A 7 3487 132 T D T R I N S A V 7 3488 181 S T C V S H I V A 7 3489 184 V S H I V A F A I 7 3490 190 F A I Y Y I P L I 7 3491 206 F G K Q A P A Y V 7 3492 211 P A Y V H T M I A 7 3493 213 Y V H T M I A N T 7 3494 221 T Y L L T S P L M 7 3495 228 L M N P V I Y S V 7 3496 236 V K T K Q I R R A 7 3497 246 I K I L H S K E T 7 3498 19 T L V T M L S I F 6 3499 28 W F N V R E I S F 6 3500 48 F F T V M E S S V 6 3501 61 A F D R F V A V S 6 3502 76 M I L T D S R I A 6 3503 82 R I A Q I G V A S 6 3504 124 P D V M K L S C T 6 3505 140 V G L T A M F S T 6 3506 153 L L L I L L S Y V 6 3507 158 L S Y V L I I R T 6 3508 159 S Y V L I I R T V 6 3509 180 F S T C V S H I V 6 3510 120 Y C Y H P D V M K 5 3511 205 R F G K Q A P A Y 5 3512 207 G K Q A P A Y V H 5 3513 227 P L M N P V I Y S 5 3514 62 F D R F V A V S N 4 3515 92 I R G L L M L T P 4 3516 133 D T R I N S A V G 4 3517 139 A V G L T A M F S 4 3518 144 A M F S T V G V D 4 3519 164 I R T V L S V A S 4 3520 169 S V A S P E E R K 4 3521 191 A I Y Y I P L I S 4 3522 197 L I S L S I V H R 4 3523 4 F L S M L S A T D 3 3524 10 A T D L G L S I S 3 3525 33 E I S F N A C L S 3 3526 34 I S F N A C L S H 3 3527 39 C L S H M F F I K 3 3528 45 F I K F F T V M E 3 3529 72 L R Y A M I L T D 3 3530 73 R Y A M I L T D S 3 3531 77 I L T D S R I A Q 3 3532 85 Q I G V A S V I R 3 3533 86 I G V A S V I R Q 3 3534 102 V A L L I R L S Y 3 3535 110 Y C H S Q V L H H 3 3536 117 H H S Y C Y H P D 3 3537 142 L T A M F S T V G 3 3538 161 V L I I R T V L S 3 3539 165 R T V L S V A S P 3 3540 167 V L S V A S P E E 3 3541 170 V A S P E E R K E 3 3542 177 K E T F S T C V S 3 3543 178 E T F S T C V S H 3 3544 199 S L S I V H R F G 3 3545 203 V H R F G K Q A P 3 3546 212 A Y V H T M I A N 3 3547 218 I A N T Y L L I S 3 3548 223 L L I S P L M N P 3 3549 239 K Q I R R A V I K 3 3550 243 R A V I K I L H S 3 3551 244 A V I K I L H S K 3 3552 17 I S T L V T M L S 2 3553 22 T M L S I F W F N 2 3554 24 L S I F W F N V R 2 3555 25 S I F W F N V R E 2 3556 31 V R E I S F N A C 2 3557 46 I K F F T V M E S 2 3558 55 S V L L A M A F D 2 3559 65 F V A V S N P L R 2 3560 66 V A V S N P L R Y 2 3561 74 Y A M I L T D S R 2 3562 79 T D S R I A Q I G 2 3563 103 A L L I R L S Y C 2 3564 105 L I R L S Y C H S 2 3565 109 S Y C H S Q V L H 2 3566 111 C H S Q V L H H S 2 3567 125 D V M K L S C T D 2 3568 151 V D L L L I L L S 2 3569 152 D L L L I L L S Y 2 3570 157 L L S Y V L I I R 2 3571 173 P E E R K E T F S 2 3572 175 E R K E T F S T C 2 3573 185 S H I V A F A I Y 2 3574 219 A N T Y L L I S P 2 3575 222 Y L L I S P L M N 2 3576 242 R R A V I K I L H 2 3577 6 S M L S A T D L G 1 3578 8 L S A T D L G L S 1 3579 20 L V T M L S I F W 1 3580 27 F W F N V R E I S 1 3581 29 F N V R E I S F N 1 3582 47 K F F T V M E S S 1 3583 56 V L L A M A F D R 1 3584 63 D R F V A V S N P 1 3585 101 M V A L L I R L S 1 3586 106 I R L S Y C H S Q 1 3587 127 M K L S C T D T R 1 3588 129 L S C T D T R I N 1 3589 130 S C T D T R I N S 1 3590 166 T V L S V A S P E 1 3591 168 L S V A S P E E R 1 3592 186 H I V A F A I Y Y 1 3593 188 V A F A I Y Y I P 1 3594 193 Y Y I P L I S L S 1 3595 196 P L I S L S I V H 1 3596 200 L S I V H R F G K 1 3597 201 S I V H R F G K Q 1 3598 229 M N P V I Y S V K 1 3599 231 P V I Y S V K T K 1 3600 232 V I Y S V K T K Q 1 3601 234 Y S V K T K Q I R 1 3602 HLA-B*08 9-mers v.1: 238P1B2 172 S P E E R K E T F 24 3603 235 S V K T K Q I R R 22 3604 233 I Y S V K T K Q I 19 3605 241 I R R A V I K I L 19 3606 12 D L G L S I S T L 18 3607 16 S I S T L V T M L 17 3608 69 S N P L R Y A M I 17 3609 97 M L T P M V A L L 17 3610 154 L L I L L S Y V L 17 3611 240 Q I R R A V I K I 17 3612 7 M L S A T D L G L 16 3613 45 F I K F F T V M E 16 3614 70 N P L R Y A M I L 16 3615 88 V A S V I R G L L 16 3616 161 V L I I R T V L S 16 3617 28 W F N V R E I S F 15 3618 78 L T D S R I A Q I 15 3619 243 R A V I K I L H S 15 3620 245 V I K I L H S K E 15 3621 60 M A F D R F V A V 14 3622 103 A L L I R L S Y C 14 3623 156 I L L S Y V L I I 14 3624 175 E R K E T F S T C 14 3625 201 S I V H R F G K Q 14 3626 238 T K Q I R R A V I 14 3627 50 T V M E S S V L L 13 3628 71 P L R Y A M I L T 13 3629 146 F S T V G V D L L 13 3630 190 F A I Y Y I P L I 13 3631 210 A P A Y V H T M I 13 3632 9 S A T D L G L S I 12 3633 19 T L V T M L S I F 12 3634 57 L L A M A F D R F 12 3635 87 G V A S V I R G L 12 3636 90 S V I R G L L M L 12 3637 96 L M L T P M V A L 12 3638 126 V M K L S C T D T 12 3639 128 K L S C T D T R I 12 3640 138 S A V G L T A M F 12 3641 147 S T V G V D L L L 12 3642 150 G V D L L L I L L 12 3643 155 L I L L S Y V L I 12 3644 215 H T M I A N T Y L 12 3645 220 N T Y L L I S P L 12 3646 5 L S M L S A T D L 11 3647 32 R E I S F N A C L 11 3648 37 N A C L S H M F F 11 3649 49 F T V M E S S V L 11 3650 91 V I R G L L M L T 11 3651 105 L I R L S Y C H S 11 3652 121 C Y H P D V M K L 11 3653 134 T R I N S A V G L 11 3654 145 M F S T V G V D L 11 3655 149 V G V D L L L I L 11 3656 163 I I R T V L S V A 11 3657 192 I Y Y I P L I S L 11 3658 194 Y I P L I S L S I 11 3659 204 H R F G K Q A P A 11 3660 206 F G K Q A P A Y V 11 3661 237 K T K Q I R R A V 11 3662 41 S H M F F I K F F 10 3663 43 M F F I K F F T V 10 3664 54 S S V L L A M A F 10 3665 64 R F V A V S N P L 10 3666 100 P M V A L L I R L 10 3667 108 L S Y C H S Q V L 10 3668 124 P D V M K L S C T 10 3669 160 Y V L I I R T V L 10 3670 173 P E E R K E T F S 10 3671 189 A F A I Y Y I P L 10 3672 216 T M I A N T Y L L 10 3673 217 M I A N T Y L L I 10 3674 18 S T L V T M L S I 9 3675 21 V T M L S I F W F 9 3676 26 I F W F N V R E I 9 3677 131 C T D T R I N S A 9 3678 167 V L S V A S P E E 9 3679 182 T C V S H I V A F 9 3680 225 I S P L M N P V I 9 3681 226 S P L M N P V I Y 9 3682 239 K Q I R R A V I K 9 3683 25 S I F W F N V R E 8 3684 30 N V R E I S F N A 8 3685 62 F D R F V A V S N 8 3686 77 I L T D S R I A Q 8 3687 89 A S V I R G L L M 8 3688 123 H P D V M K L S C 8 3689 174 E E R K E T F S T 8 3690 184 V S H I V A F A I 8 3691 187 I V A F A I Y Y I 8 3692 198 I S L S I V H R F 8 3693 199 S L S I V H R F G 8 3694 203 V H R F G K Q A P 8 3695 4 F L S M L S A T D 7 3696 14 G L S I S T L V T 7 3697 36 F N A C L S H M F 7 3698 56 V L L A M A F D R 7 3699 75 A M I L T D S R I 7 3700 94 G L L M L T P M V 7 3701 95 L L M L T P M V A 7 3702 99 T P M V A L L I R 7 3703 107 R L S Y C H S Q V 7 3704 115 V L H H S Y C Y H 7 3705 141 G L T A M F S T V 7 3706 148 T V G V D L L L I 7 3707 152 D L L L I L L S Y 7 3708 157 L L S Y V L I I R 7 3709 179 T F S T C V S H I 7 3710 195 I P L I S L S I V 7 3711 197 L I S L S I V H R 7 3712 227 P L M N P V I Y S 7 3713 230 N P V I Y S V K T 7 3714 23 M L S I F W F N V 6 3715 33 E I S F N A C L S 6 3716 38 A C L S H M F F I 6 3717 39 C L S H M F F I K 6 3718 40 L S H M F F I K F 6 3719 80 D S R I A Q I G V 6 3720 82 R I A Q I G V A S 6 3721 83 I A Q I G V A S V 6 3722 84 A Q I G V A S V I 6 3723 85 Q I G V A S V I R 6 3724 98 L T P M V A L L I 6 3725 102 V A L L I R L S Y 6 3726 104 L L I R L S Y C H 6 3727 133 D T R I N S A V G 6 3728 153 L L L I L L S Y V 6 3729 196 P L I S L S I V H 6 3730 222 Y L L I S P L M N 6 3731 223 L L I S P L M N P 6 3732 66 V A V S N P L R Y 5 3733 135 R I N S A V G L T 5 3734 143 T A M F S T V G V 5 3735 162 L I I R T V L S V 5 3736 170 V A S P E E R K E 5 3737 186 H I V A F A I Y Y 5 3738 188 V A F A I Y Y I P 5 3739 191 A I Y Y I P L I S 5 3740 209 Q A P A Y V H T M 5 3741 211 P A Y V H T M I A 5 3742 218 I A N T Y L L I S 5 3743 232 V I Y S V K T K Q 5 3744 58 L A M A F D R F V 4 3745 74 Y A M I L T D S R 4 3746 76 M I L T D S R I A 4 3747 159 S Y V L I I R T V 4 3748 164 I R T V L S V A S 4 3749 171 A S P E E R K E T 4 3750 224 L I S P L M N P V 4 3751 246 I K I L H S K E T 4 3752 46 I K F F T V M E S 3 3753 53 E S S V L L A M A 3 3754 55 S V L L A M A F D 3 3755 65 F V A V S N P L R 3 3756 81 S R I A Q I G V A 3 3757 109 S Y C H S Q V L H 3 3758 113 S Q V L H H S Y C 3 3759 119 S Y C Y H P D V M 3 3760 151 V D L L L I L L S 3 3761 178 E T F S T C V S H 3 3762 181 S T C V S H I V A 3 3763 185 S H I V A F A I Y 3 3764 231 P V I Y S V K T K 3 3765 6 S M L S A T D L G 2 3766 17 I S T L V T M L S 2 3767 29 F N V R E I S F N 2 3768 31 V R E I S F N A C 2 3769 35 S F N A C L S H M 2 3770 48 F F T V M E S S V 2 3771 51 V M E S S V L L A 2 3772 63 D R F V A V S N P 2 3773 68 V S N P L R Y A M 2 3774 86 I G V A S V I R G 2 3775 92 I R G L L M L T P 2 3776 101 M V A L L I R L S 2 3777 112 H S Q V L H H S Y 2 3778 117 H H S Y C Y H P D 2 3779 130 S C T D T R I N S 2 3780 132 T D T R I N S A V 2 3781 137 N S A V Q L T A M 2 3782 158 L S Y V L I I R T 2 3783 165 R T V L S V A S P 2 3784 169 S V A S P E E R K 2 3785 202 I V H R F G K Q A 2 3786 236 V K T K Q I R R A 2 3787 2 Y Y F L S M L S A 1 3788 3 Y F L S M L S A T 1 3789 10 A T D L G L S I S 1 3790 15 L S I S T L V T M 1 3791 27 F W F N V R E I S 1 3792 34 I S F N A C L S H 1 3793 42 H M F F I K F F T 1 3794 44 F F I K F F T V M 1 3795 52 M E S S V L L A M 1 3796 61 A F D R F V A V S 1 3797 72 L R Y A M I L T D 1 3798 93 R G L L M L T P M 1 3799 106 I R L S Y C H S Q 1 3800 110 Y C H S Q V L H H 1 3801 118 H S Y C Y H P D V 1 3802 120 Y C Y H P D V M K 1 3803 122 Y H P D V M K L S 1 3804 136 I N S A V G L T A 1 3805 139 A V G L T A M F S 1 3806 140 V G L T A M F S T 1 3807 144 A M F S T V G V D 1 3808 168 L S V A S P E E R 1 3809 180 F S T C V S H I V 1 3810 183 C V S H I V A F A 1 3811 193 Y Y I P L I S L S 1 3812 200 L S I V H R F G K 1 3813 205 R F G K Q A P A Y 1 3814 207 G K Q A P A Y V H 1 3815 208 K Q A P A Y V H T 1 3816 212 A Y V H T M I A N 1 3817 213 Y V H T M I A N T 1 3818 214 V H T M I A N T Y 1 3819 219 A N T Y L L I S P 1 3820 221 T Y L L I S P L M 1 3821 228 L M N P V I Y S V 1 3822 229 M N P V I Y S V K 1 3823 242 R R A V I K I L H 1 3824 HLA-B*1510 9-mers v.1: 238P1B2 41 S H M F F I K F F 17 3825 87 G V A S V I R G L 15 3826 96 L M L T P M V A L 15 3827 50 T V M E S S V L L 14 3828 160 Y V L I I R T V L 14 3829 88 V A S V I R G L L 13 3830 97 M L T P M V A L L 13 3831 121 C Y H P D V M K L 13 3832 122 Y H P D V M K L S 13 3833 134 T R I N S A V G L 13 3834 145 M F S T V G V D L 13 3835 146 F S T V G V D L L 13 3836 192 I Y Y I P L I S L 13 3837 203 V H R F G K Q A P 13 3838 241 I R R A V I K I L 13 3839 12 D L G L S I S T L 12 3840 16 S I S T L V T M L 12 3841 49 F T V M E S S V L 12 3842 100 P M V A L L I R L 12 3843 108 L S Y C H S Q V L 12 3844 111 C H S Q V L H H S 12 3845 154 L L I L L S Y V L 12 3846 216 T M I A N T Y L L 12 3847 7 M L S A T D L G L 11 3848 90 S V I R G L L M L 11 3849 117 H H S Y C Y H P D 11 3850 147 S T V G V D L L L 11 3851 149 V G V D L L L I L 11 3852 150 G V D L L L I L L 11 3853 182 T C V S H I V A F 11 3854 185 S H I V A F A I Y 11 3855 189 A F A I Y Y I P L 11 3856 198 I S L S I V H R F 11 3857 214 V H T M I A N T Y 11 3858 220 N T Y L L I S P L 11 3859 5 L S M L S A T D L 10 3860 32 R E I S F N A C L 10 3861 64 R F V A V S N P L 10 3862 68 V S N P L R Y A M 10 3863 70 N P L R Y A M I L 10 3864 116 L H H S Y C Y H P 10 3865 137 N S A V G L T A M 10 3866 172 S P E E R K E T F 10 3867 215 H T M I A N T Y L 10 3868 15 L S I S T L V T M 9 3869 44 F F I K F F T V M 9 3870 57 L L A M A F D R F 9 3871 119 S Y C Y H P D V M 9 3872 52 M E S S V L L A M 8 3873 209 Q A P A Y V H T M 8 3874 221 T Y L L I S P L M 8 3875 19 T L V T M L S I F 7 3876 21 V T M L S I F W F 7 3877 26 I F W F N V R E I 7 3878 28 W F N V R E I S F 7 3879 36 F N A C L S H M F 7 3880 54 S S V L L A M A F 7 3881 138 S A V G L T A M F 7 3882 35 S F N A C L S H M 6 3883 37 N A C L S H M F F 6 3884 40 L S H M F F I K F 6 3885 89 A S V I R G L L M 6 3886 93 R G L L M L T P M 6 3887 164 I R T V L S V A S 6 3888 236 V K T K Q I R R A 6 3889 77 I L T D S R I A Q 5 3890 82 R I A Q I G V A S 5 3891 86 I G V A S V I R G 5 3892 170 V A S P E E R K E 5 3893 208 K Q A P A Y V H T 5 3894 225 I S P L M N P V I 5 3895 237 K T K Q I R R A V 5 3896 14 G L S I S T L V T 4 3897 25 S I F W F N V R E 4 3898 46 I K F F T V M E S 4 3899 59 A M A F D R F V A 4 3900 60 M A F D R F V A V 4 3901 66 V A V S N P L R Y 4 3902 67 A V S N P L R Y A 4 3903 83 I A Q I G V A S V 4 3904 92 I R G L L M L T P 4 3905 101 M V A L L I R L S 4 3906 120 Y C Y H P D V M K 4 3907 136 I N S A V G L T A 4 3908 158 L S Y V L I I R T 4 3909 159 S Y V L I I R T V 4 3910 163 I I R T V L S V A 4 3911 167 V L S V A S P E E 4 3912 169 S V A S P E E R K 4 3913 187 I V A F A I Y Y I 4 3914 193 Y Y I P L I S L S 4 3915 197 L I S L S I V H R 4 3916 207 G K Q A P A Y V H 4 3917 224 L I S P L M N P V 4 3918 226 S P L M N P V I Y 4 3919 233 I Y S V K T K Q I 4 3920 238 T K Q I R R A V I 4 3921 33 E I S F N A C L S 3 3922 53 E S S V L L A M A 3 3923 61 A F D R F V A V S 3 3924 65 F V A V S N P L R 3 3925 81 S R I A Q I G V A 3 3926 84 A Q I G V A S V I 3 3927 85 Q I G V A S V I R 3 3928 95 L L M L T P M V A 3 3929 128 K L S C T D T R I 3 3930 133 D T R I N S A V G 3 3931 142 L T A M F S T V G 3 3932 143 T A M F S T V G V 3 3933 144 A M F S T V G V D 3 3934 156 I L L S Y V L I I 3 3935 171 A S P E E R K E T 3 3936 175 E R K E T F S T C 3 3937 178 E T F S T C V S H 3 3938 179 T F S T C V S H I 3 3939 181 S T C V S H I V A 3 3940 199 S L S I V H R F G 3 3941 205 R F G K Q A P A Y 3 3942 228 L M N P V I Y S V 3 3943 229 M N P V I Y S V K 3 3944 234 Y S V K T K Q I R 3 3945 242 R R A V I K I L H 3 3946 246 I K I L H S K E T 3 3947 2 Y Y F L S M L S A 2 3948 3 Y F L S M L S A T 2 3949 4 F L S M L S A T D 2 3950 11 T D L G L S I S T 2 3951 17 I S T L V T M L S 2 3952 18 S T L V T M L S I 2 3953 24 L S T F W F N V R 2 3954 31 V R E I S F N A C 2 3955 34 I S F N A C L S H 2 3956 45 F I K F F T V M E 2 3957 51 V M E S S V L L A 2 3958 58 L A M A F D R F V 2 3959 62 F D R F V A V S N 2 3960 63 D R F V A V S N P 2 3961 72 L R Y A M I L T D 2 3962 75 A M I L T D S R I 2 3963 76 M I L T D S R I A 2 3964 79 T D S R I A Q I G 2 3965 94 G L L M L T P M V 2 3966 99 T P M V A L L I R 2 3967 106 I R L S Y C H S Q 2 3968 109 S Y C H S Q V L H 2 3969 110 Y C H S Q V L H H 2 3970 123 H P D V M K L S C 2 3971 125 D V M K L S C T D 2 3972 129 L S C T D T R I N 2 3973 130 S C T D T R I N S 2 3974 132 T D T R I N S A V 2 3975 141 G L T A M F S T V 2 3976 148 T V G V D L L L I 2 3977 155 L I L L S Y V L I 2 3978 161 V L I I R T V L S 2 3979 168 L S V A S P E E R 2 3980 174 E E R K E T F S T 2 3981 177 K E T F S T C V S 2 3982 183 C V S H I V A F A 2 3983 190 F A I Y Y I P L I 2 3984 195 I P L I S L S I V 2 3985 196 P L I S L S I V H 2 3986 200 L S I V H R F G K 2 3987 202 T V H R F G K Q A 2 3988 206 F G K Q A P A Y V 2 3989 212 A Y V H T M I A N 2 3990 213 Y V H T M I A N T 2 3991 218 I A N T Y L L I S 2 3992 222 Y L L I S P L M N 2 3993 227 P L M N P V I Y S 2 3994 230 N P V I Y S V K T 2 3995 231 P V I Y S V K T K 2 3996 235 S V K T K Q I R R 2 3997 239 K Q I R R A V I K 2 3998 8 L S A T D L G L S 1 3999 9 S A T D L G L S I 1 4000 10 A T D L G L S I S 1 4001 22 T M L S I F W F N 1 4002 23 M L S I E W F N V 1 4003 27 F W E N V R E I S 1 4004 29 F N V R E I S E N 1 4005 30 N V R E I S F N A 1 4006 38 A C L S H M F F I 1 4007 39 C L S H M F F I K 1 4008 42 H M F F I K F F T 1 4009 43 M F F I K F F T V 1 4010 47 K F F T V M E S S 1 4011 48 F F T V M E S S V 1 4012 55 S V L L A M A F D 1 4013 71 P L R Y A M I L T 1 4014 73 R Y A M I L T D S 1 4015 74 Y A M I L T D S R 1 4016 78 L T D S R I A Q I 1 4017 91 V I R G L L M L T 1 4018 102 V A L L I R L S Y 1 4019 107 R L S Y C H S Q V 1 4020 112 H S Q V L H H S Y 1 4021 118 H S Y C Y H P D V 1 4022 127 M K L S C T D T R 1 4023 131 C T D T R I N S A 1 4024 135 R I N S A V G L T 1 4025 140 V G L T A M F S T 1 4026 151 V D L L L I L L S 1 4027 157 L L S Y V L I I R 1 4028 162 L I I R T V L S V 1 4029 165 R T V L S V A S P 1 4030 166 T V L S V A S P E 1 4031 173 P E E R K E T F S 1 4032 176 R K E T F S T C V 1 4033 184 V S H I V A F A I 1 4034 191 A I Y Y I P L I S 1 4035 194 Y I P L I S L S I 1 4036 204 H R F G K Q A P A 1 4037 210 A P A Y V H T M I 1 4038 211 P A Y V H T M I A 1 4039 217 M I A N T Y L L I 1 4040 223 L L I S P L M N P 1 4041 232 V I Y S V K T K Q 1 4042 240 Q I R R A V I K I 1 4043 243 R A V I K I L H S 1 4044 244 A V I K I L H S K 1 4045 HLA-B*2705 9-mers v.1: 238P1B2 134 T R I N S A V G L 25 4046 241 I R R A V I K I L 25 4047 242 R R A V I K I L H 25 4048 204 H R F G K Q A P A 19 4049 32 R E I S F N A C L 18 4050 64 R F V A V S N P L 18 4051 93 R G L L M L T P M 18 4052 220 N T Y L L I S P L 18 4053 244 A V I K I L H S K 18 4054 12 D L G L S I S T L 17 4055 63 D R F V A V S N P 17 4056 72 L R Y A M I L T D 17 4057 87 G V A S V I R G L 17 4058 150 G V D L L L I L L 17 4059 154 L L I L L S Y V L 17 4060 192 I Y Y I P L I S L 17 4061 198 I S L S I V H R F 17 4062 81 S R I A Q I G V A 16 4063 90 S V I R G L L M L 16 4064 100 P M V A L L I R L 16 4065 147 S T V G V D L L L 16 4066 160 Y V L I I R T V L 16 4067 197 L I S L S I V H R 16 4068 205 R F G K Q A P A Y 16 4069 235 S V K T K Q I R R 16 4070 239 K Q I R R A V I K 16 4071 5 L S M L S A T D L 15 4072 21 V T M L S I F W F 15 4073 44 F F I K F F T V M 15 4074 49 F T V M E S S V L 15 4075 54 S S V L L A M A F 15 4076 84 A Q I G V A S V I 15 4077 92 I R G L L M L T P 15 4078 120 Y C Y H P D V M K 15 4079 121 C Y R P D V M K L 15 4080 138 S A V G L T A M F 15 4081 152 D L L L I L L S Y 15 4082 182 T C V S H I V A F 15 4083 207 G K Q A P A Y V H 15 4084 231 P V I Y S V K T K 15 4085 16 S I S T L V T M L 14 4086 24 L S I F W F N V R 14 4087 34 I S F N A C L S H 14 4088 50 T V M E S S V L L 14 4089 70 N P L R Y A M I L 14 4090 75 A M I L T D S R I 14 4091 96 L M L T P M V A L 14 4092 102 V A L L I R L S Y 14 4093 106 I R L S Y C H S Q 14 4094 108 L S Y C H S Q V L 14 4095 149 V G V D L L L I L 14 4096 164 I R T V L S V A S 14 4097 196 P L I S L S I V H 14 4098 221 T Y L L I S P L M 14 4099 234 Y S V K T K Q I R 14 4100 15 L S I S T L V T M 13 4101 19 T L V T M L S I F 13 4102 37 N A C L S H M F F 13 4103 40 L S H M F F I K F 13 4104 41 S H M F F I K F F 13 4105 56 V L L A M A F D R 13 4106 65 F V A V S N P L R 13 4107 97 M L T P M V A L L 13 4108 104 L L I R L S Y C H 13 4109 127 M K L S C T D T R 13 4110 128 K L S C T D T R I 13 4111 137 N S A V G L T A M 13 4112 146 F S T V G V D L L 13 4113 157 L L S Y V L I I R 13 4114 168 L S V A S P E E R 13 4115 169 S V A S P E E R K 13 4116 172 S P E E R K E T F 13 4117 175 E R K E T F S T C 13 4118 178 E T F S T C V S H 13 4119 215 H T M I A N T Y L 13 4120 216 T M I A N T Y L L 13 4121 229 M N P V I Y S V K 13 4122 7 M L S A T D L G L 12 4123 28 W F N V R E I S F 12 4124 31 V R E I S F N A C 12 4125 36 F N A C L S H M F 12 4126 57 L L A M A F D R F 12 4127 66 V A V S N P L R Y 12 4128 74 Y A M I L T D S R 12 4129 85 Q I G V A S V I R 12 4130 99 T P M V A L L I R 12 4131 109 S Y C H S Q V L H 12 4132 114 Q V L H H S Y C Y 12 4133 145 M F S T V G V D L 12 4134 189 A F A I Y Y I P L 12 4135 190 F A I Y Y I P L I 12 4136 200 L S I V H R F G K 12 4137 214 V H T M I A N T Y 12 4138 52 M E S S V L L A M 11 4139 68 V S N P L R Y A M 11 4140 78 L T D S R I A Q I 11 4141 88 V A S V I R G L L 11 4142 89 A S V I R G L L M 11 4143 155 L I L L S Y V L I 11 4144 156 I L L S Y V L I I 11 4145 185 S H I V A F A I Y 11 4146 186 H I V A F A I Y Y 11 4147 187 I V A F A I Y Y I 11 4148 194 Y I P L I S L S I 11 4149 225 I S P L M N P V I 11 4150 226 S P L M N P V I Y 11 4151 18 S T L V T M L S I 10 4152 35 S F N A C L S H M 10 4153 39 C L S H M F F I K 10 4154 110 Y C H S Q V L H H 10 4155 112 H S Q V L H H S Y 10 4156 165 R T V L S V A S P 10 4157 209 Q A P A Y V H T M 10 4158 210 A P A Y V H T M I 10 4159 240 Q I R R A V I K I 10 4160 243 R A V I K I L H S 10 4161 9 S A T D L G L S I 9 4162 26 I F W F N V R E I 9 4163 38 A C L S H M F F I 9 4164 98 L T P M V A L L I 9 4165 115 V L H H S Y C Y H 9 4166 119 S Y C Y H P D V M 9 4167 158 L S Y V L I I R T 9 4168 233 I Y S V K T K Q I 9 4169 11 T D L G L S I S T 8 4170 69 S N P L R Y A M I 8 4171 73 R Y A M I L T D S 8 4172 82 R I A Q I G V A S 8 4173 148 T V G V D L L L I 8 4174 151 V D L L L I L L S 8 4175 179 T F S T C V S H I 8 4176 184 V S H I V A F A I 8 4177 193 Y Y I P L I S L S 8 4178 232 V I Y S V K T K Q 8 4179 238 T K Q I R R A V I 8 4180 2 Y Y F L S M L S A 7 4181 25 S I F W F N V R E 7 4182 46 I K F F T V M E S 7 4183 47 K F F T V M E S S 7 4184 86 I G V A S V I R G 7 4185 94 G L L M L T P M V 7 4186 159 S Y V L I I R T V 7 4187 176 R K E T F S T C V 7 4188 217 M I A N T Y L L I 7 4189 223 L L I S P L M N P 7 4190 228 L M N P V I Y S V 7 4191 236 V K T K Q I R R A 7 4192 10 A T D L G L S I S 6 4193 13 L G L S I S T L V 6 4194 14 G L S I S T L V T 6 4195 29 F N V R E I S F N 6 4196 83 I A Q I G V A S V 6 4197 107 R L S Y C H S Q V 6 4198 144 A M F S T V G V D 6 4199 195 I P L I S L S I V 6 4200 213 Y V H T M I A N T 6 4201 246 I K I L H S K E T 6 4202 1 M Y Y F L S M L S 5 4203 30 N V R E I S F N A 5 4204 42 H M F F I K F F T 5 4205 43 M F F I K F F T V 5 4206 91 V I R G L L M L T 5 4207 101 M V A L L I R L S 5 4208 103 A L L I R L S Y C 5 4209 125 D V M K L S C T D 5 4210 131 C T D T R I N S A 5 4211 135 R I N S A V G L T 5 4212 141 G L T A M F S T V 5 4213 153 L L L I L L S Y V 5 4214 162 L I I R T V L S V 5 4215 163 I I R T V L S V A 5 4216 166 T V L S V A S P E 5 4217 188 V A F A I Y Y I P 5 4218 191 A I Y Y I P L I S 5 4219 219 A N T Y L L I S P 5 4220 230 N P V I Y S V K T 5 4221 237 K T K Q I R R A V 5 4222 3 Y F L S M L S A T 4 4223 4 F L S M L S A T D 4 4224 17 I S T L V T M L S 4 4225 22 T M L S I F W F N 4 4226 27 F W F N V R E I S 4 4227 55 S V L L A M A F D 4 4228 60 M A F D R F V A V 4 4229 61 A F D R F V A V S 4 4230 62 F D R F V A V S N 4 4231 111 C H S Q V L H H S 4 4232 124 P D V M K L S C T 4 4233 126 V M K L S C T D T 4 4234 136 I N S A V G L T A 4 4235 139 A V G L T A M F S 4 4236 142 L T A M F S T V G 4 4237 170 V A S P E E R K E 4 4238 171 A S P E E R K E T 4 4239 174 E E R K E T F S T 4 4240 177 K E T F S T C V S 4 4241 208 K Q A P A Y V H T 4 4242 222 Y L L I S P L M N 4 4243 245 V I K I L H S K E 4 4244 20 L V T M L S I F W 3 4245 51 V M E S S V L L A 3 4246 53 E S S V L L A M A 3 4247 67 A V S N P L R Y A 3 4248 76 M I L T D S R I A 3 4249 77 I L T D S R I A Q 3 4250 80 D S R I A Q I G V 3 4251 95 L L M L T P M V A 3 4252 113 S Q V L H H S Y C 3 4253 118 H S Y C Y H P D V 3 4254 123 H P D V M K L S C 3 4255 130 S C T D T R I N S 3 4256 132 T D T R I N S A V 3 4257 133 D T R I N S A V G 3 4258 140 V G L T A M F S T 3 4259 161 V L I I R T V L S 3 4260 167 V L S V A S P E E 3 4261 173 P E E R K E T F S 3 4262 181 S T C V S H I V A 3 4263 183 C V S H I V A F A 3 4264 201 S I V H R F G K Q 3 4265 203 V H R F G K Q A P 3 4266 206 F G K Q A P A Y V 3 4267 211 P A Y V H T M I A 3 4268 212 A Y V H T M I A N 3 4269 218 I A N T Y L L I S 3 4270 224 L I S P L M N P V 3 4271 227 P L M N P V I Y S 3 4272 6 S M L S A T D L G 2 4273 23 M L S I F W F N V 2 4274 48 F F T V M E S S V 2 4275 59 A M A F D R F V A 2 4276 79 T D S R I A Q I G 2 4277 105 L I R L S Y C H S 2 4278 143 T A M F S T V G V 2 4279 180 F S T C V S H I V 2 4280 202 I V H R F G K Q A 2 4281 8 L S A T D L G L S 1 4282 33 E I S F N A C L S 1 4283 45 E I K E F T V M E 1 4284 58 L A M A F D R F V 1 4285 71 P L R Y A M I L T 1 4286 116 L H H S Y C Y H P 1 4287 122 Y H P D V M K L S 1 4288 199 S L S I V H R F G 1 4289 HLA-B*2709 9-mers v.1: 238P1B2 134 T R I N S A V G L 23 4290 241 I R R A V I K I L 21 4291 32 R E I S F N A C L 16 4292 64 R F V A V S N P L 15 4293 198 I S L S I V H R F 15 4294 242 R R A V I K I L H 15 4295 63 D R F V A V S N P 14 4296 87 G V A S V I R G L 14 4297 93 R G L L M L T P M 14 4298 94 G L L M L T P M V 14 4299 106 I R L S Y C H S Q 14 4300 156 I L L S Y V L I I 14 4301 70 N P L R Y A M I L 13 4302 72 L R Y A M I L T D 13 4303 96 L M L T P M V A L 13 4304 97 M L T P M V A L L 13 4305 107 R L S Y C H S Q V 13 4306 121 C Y H P D V M K L 13 4307 150 G V D L L L I L L 13 4308 160 Y V L I I R T V L 13 4309 164 I R T V L S V A S 13 4310 192 I Y Y I P L I S L 13 4311 13 L G L S I S T L V 12 4312 38 A C L S H M F F I 12 4313 90 S V I R G L L M L 12 4314 92 I R G L L M L T P 12 4315 100 P M V A L L I R L 12 4316 108 L S Y C H S Q V L 12 4317 141 G L T A M F S T V 12 4318 146 F S T V G V D L L 12 4319 147 S T V G V D L L L 12 4320 149 V G V D L L L I L 12 4321 154 L L I L L S Y V L 12 4322 176 R K E T F S T C V 12 4323 204 H R F G K Q A P A 12 4324 216 T M I A N T Y L L 12 4325 220 N T Y L L I S P L 12 4326 221 T Y L L I S P L M 12 4327 5 L S M L S A T D L 11 4328 7 M L S A T D L G L 11 4329 15 L S I S T L V T M 11 4330 16 S I S T L V T M L 11 4331 18 S T L V T M L S I 11 4332 49 F T V M E S S V L 11 4333 50 T V M E S S V L L 11 4334 60 M A F D R F V A V 11 4335 75 A M I L T D S R I 11 4336 81 S R I A Q I G V A 11 4337 84 A Q I G V A S V I 11 4338 88 V A S V I R G L L 11 4339 89 A S V I R G L L M 11 4340 118 H S Y C Y H P D V 11 4341 128 K L S C T D T R I 11 4342 145 M F S T V G V D L 11 4343 155 L I L L S Y V L I 11 4344 162 L I I R T V L S V 11 4345 182 T C V S H I V A F 11 4346 189 A F A I Y Y I P L 11 4347 195 I P L I S L S I V 11 4348 237 K T K Q I R R A V 11 4349 9 S A T D L G L S I 10 4350 12 D L G L S I S T L 10 4351 26 I F W F N V R E I 10 4352 31 V R E I S F N A C 10 4353 43 M F F I K F F T V 10 4354 52 M E S S V L L A M 10 4355 54 S S V L L A M A F 10 4356 68 V S N P L R Y A M 10 4357 78 L T D S R I A Q I 10 4358 83 I A Q I G V A S V 10 4359 98 L T P M V A L L I 10 4360 132 T D T R I N S A V 10 4361 148 T V G V D L L L I 10 4362 153 L L L I L L S Y V 10 4363 159 S Y V L I I R T V 10 4364 175 E R K E T F S T C 10 4365 187 I V A F A I Y Y I 10 4366 190 F A I Y Y I P L I 10 4367 194 Y I P L I S L S I 10 4368 215 H T M I A N T Y L 10 4369 225 I S P L M N P V I 10 4370 228 L M N P V I Y S V 10 4371 233 I Y S V K T K Q I 10 4372 19 T L V T M L S I F 9 4373 23 M L S I F W F N V 9 4374 44 F F I K F F T V M 9 4375 48 F F T V M E S S V 9 4376 57 L L A M A F D R F 9 4377 138 S A V G L T A M F 9 4378 143 T A M F S T V G V 9 4379 179 T F S T C V S H I 9 4380 180 F S T C V S H I V 9 4381 184 V S H I V A F A I 9 4382 209 Q A P A Y V H T M 9 4383 210 A P A Y V H T M I 9 4384 217 M I A N T Y L L I 9 4385 224 L I S P L M N P V 9 4386 240 Q I R R A V I K I 9 4387 21 V T M L S I F W F 8 4388 28 W F N V R E I S F 8 4389 35 S F N A C L S H M 8 4390 36 F N A C L S H M F 8 4391 37 N A C L S H M F F 8 4392 40 L S H M F F I K F 8 4393 41 S H M F F I K F F 8 4394 58 L A M A F D R F V 8 4395 69 S N P L R Y A M I 8 4396 80 D S R I A Q I G V 8 4397 119 S Y C Y H P D V M 8 4398 137 N S A V G L T A M 8 4399 172 S P E E R K E T F 8 4400 206 F G K Q A P A Y V 8 4401 238 T K Q I R R A V I 8 4402 82 R I A Q I G V A S 6 4403 135 R I N S A V G L T 6 4404 165 R T V L S V A S P 6 4405 205 R F G K Q A P A Y 6 4406 243 R A V I K I L H S 6 4407 73 R Y A M I L T D S 5 4408 144 A M F S T V G V D 5 4409 191 A I Y Y I P L I S 5 4410 207 G K Q A P A Y V H 5 4411 239 K Q I R R A V I K 5 4412 14 G L S I S T L V T 4 4413 22 T M L S I F W F N 4 4414 25 S I F W F N V R E 4 4415 34 I S F N A C L S H 4 4416 46 I K F F T V M E S 4 4417 47 K F F T V M E S S 4 4418 66 V A V S N P L R Y 4 4419 86 I G V A S V I R G 4 4420 102 V A L L I R L S Y 4 4421 152 D L L L I L L S Y 4 4422 166 T V L S V A S P E 4 4423 178 E T F S T G V S H 4 4424 188 V A F A T Y Y I P 4 4425 208 K Q A P A Y V H T 4 4426 2 Y Y F L S M L S A 3 4427 3 Y F L S M L S A T 3 4428 6 S M L S A T D L G 3 4429 42 H M F F I K F F T 3 4430 51 V M E S S V L L A 3 4431 56 V L L A M A F D R 3 4432 76 M I L T D S R I A 3 4433 103 A L L I R L S Y C 3 4434 110 Y C H S Q V L H H 3 4435 114 Q V L H H S Y C Y 3 4436 120 Y C Y H P D V M K 3 4437 127 M K L S C T D T R 3 4438 151 V D L L L I L L S 3 4439 158 L S Y V L I I R T 3 4440 177 K E T F S T C V S 3 4441 214 V H T M I A N T Y 3 4442 222 Y L L I S P L M N 3 4443 223 L L I S P L M N P 3 4444 226 S P L M N P V I Y 3 4445 232 V I Y S V K T K Q 3 4446 244 A V I K I L H S K 3 4447 1 M Y Y F L S M L S 2 4448 11 T D L G L S I S T 2 4449 17 I S T L V T M L S 2 4450 27 F W F N V R E I S 2 4451 30 N V R E I S F N A 2 4452 55 S V L L A M A F D 2 4453 59 A M A F D R F V A 2 4454 61 A F D R F V A V S 2 4455 62 F D R F V A V S N 2 4456 77 I L T D S R I A Q 2 4457 79 T D S R I A Q I G 2 4458 104 L L I R L S Y C H 2 4459 116 L H H S Y C Y H P 2 4460 130 S C T D T R I N S 2 4461 136 I N S A V G L T A 2 4462 139 A V G L T A M F S 2 4463 140 V G L T A M F S T 2 4464 161 V L I I R T V L S 2 4465 169 S V A S P E E R K 2 4466 183 C V S H I V A F A 2 4467 186 H I V A F A I Y Y 2 4468 193 Y Y I P L I S L S 2 4469 196 P L I S L S I V H 2 4470 202 I V H R F G K Q A 2 4471 211 P A Y V H T M I A 2 4472 212 A Y V H T M I A N 2 4473 218 I A N T Y L L I S 2 4474 219 A N T Y L L I S P 2 4475 230 N P V I Y S V K T 2 4476 231 P V I Y S V K T K 2 4477 236 V K T K Q I R R A 2 4478 246 I K I L H S K E T 2 4479 8 L S A T D L G L S 1 4480 10 A T D L G L S I S 1 4481 20 L V T M L S I F W 1 4482 24 L S I F W F N V R 1 4483 29 F N V R E I S F N 1 4484 33 E I S F N A C L S 1 4485 45 F I K F F T V M E 1 4486 65 F V A V S N P L R 1 4487 67 A V S N P L R Y A 1 4488 85 Q I G V A S V I R 1 4489 99 T P M V A L L I R 1 4490 109 S Y C H S Q V L H 1 4491 113 S Q V L H H S Y C 1 4492 123 H P D V M K L S C 1 4493 124 P D V M K L S C T 1 4494 129 L S C T D T R I N 1 4495 163 I I R T V L S V A 1 4496 167 V L S V A S P E E 1 4497 168 L S V A S P E E R 1 4498 170 V A S P E E R K E 1 4499 171 A S P E E R K E T 1 4500 185 S H I V A F A I Y 1 4501 197 L I S L S I V H R 1 4502 200 L S I V H R F G K 1 4503 201 S I V H R F G K Q 1 4504 213 Y V H T M I A N T 1 4505 227 P L M N P V I Y S 1 4506 229 M N P V I Y S V K 1 4507 234 Y S V K T K Q I R 1 4508 235 S V K T K Q I R R 1 4509 HLA-B*4402 9-mers v.1: 238P1B2 32 R E I S F N A C L 23 4510 41 S H M F F I K F F 17 4511 84 A Q I G V A S V I 17 4512 90 S V I R G L L M L 17 4513 134 T R I N S A V G L 16 4514 182 T C V S H I V A F 16 4515 216 T M I A N T Y L L 16 4516 12 D L G L S I S T L 15 4517 21 V T M L S I F W F 15 4518 52 M E S S V L L A M 15 4519 54 S S V L L A M A F 15 4520 75 A M I L T D S R I 15 4521 96 L M L T P M V A L 15 4522 97 M L T P M V A L L 15 4523 102 V A L L I R L S Y 15 4524 121 C Y H P D V M K L 15 4525 150 G V D L L L I L L 15 4526 152 D L L L I L L S Y 15 4527 154 L L I L L S Y V L 15 4528 185 S H I V A F A I Y 15 4529 190 F A I Y Y I P L I 15 4530 220 N T Y L L I S P L 15 4531 226 S P L M N P V I Y 15 4532 241 I R R A V I K I L 15 4533 16 S I S T L V T M L 14 4534 70 N P L R Y A M I L 14 4535 78 L T D S R I A Q I 14 4536 87 G V A S V I R G L 14 4537 88 V A S V I R G L L 14 4538 147 S T V G V D L L L 14 4539 160 Y V L I I R T V L 14 4540 189 A F A I Y Y I P L 14 4541 198 I S L S I V H R F 14 4542 214 V H T M I A N T Y 14 4543 5 L S M L S A T D L 13 4544 40 L S H M F F I K F 13 4545 50 T V M E S S V L L 13 4546 66 V A V S N P L R Y 13 4547 138 S A V G L T A M F 13 4548 145 M F S T V G V D L 13 4549 149 V G V D L L L I L 13 4550 172 S P E E R K E T F 13 4551 174 E E R K E T F S T 13 4552 177 K E T F S T C V S 13 4553 205 R F G K Q A P A Y 13 4554 233 I Y S V K T K Q I 13 4555 7 M L S A T D L G L 12 4556 19 T L V T M L S I F 12 4557 28 W F N V R E I S F 12 4558 37 N A C L S H M F F 12 4559 38 A C L S H M F F I 12 4560 69 S N P L R Y A M I 12 4561 98 L T P M V A L L I 12 4562 100 P M V A L L I R L 12 4563 146 F S T V G V D L L 12 4564 155 L I L L S Y V L I 12 4565 186 H I V A F A I Y Y 12 4566 192 I Y Y I P L I S L 12 4567 194 Y I P L I S L S I 12 4568 240 Q I R R A V I K I 12 4569 9 S A T D L G L S I 11 4570 18 S T L V T M L S I 11 4571 20 L V T M L S I F W 11 4572 26 I F W F N V R E I 11 4573 36 F N A C L S H M F 11 4574 49 F T V M E S S V L 11 4575 57 L L A M A F D R F 11 4576 64 R F V A V S N P L 11 4577 108 L S Y C H S Q V L 11 4578 112 H S Q V L H H S Y 11 4579 114 Q V L H H S Y C Y 11 4580 148 T V G V D L L L I 11 4581 156 I L L S Y V L I I 11 4582 173 P E E R K E T F S 11 4583 179 T F S T C V S H I 11 4584 184 V S H I V A F A I 11 4585 187 I V A F A I Y Y I 11 4586 210 A P A Y V H T M I 11 4587 215 H T M I A N T Y L 11 4588 217 M I A N T Y L L I 11 4589 225 I S P L M N P V I 11 4590 238 T K Q I R R A V I 11 4591 128 K L S C T D T R I 10 4592 193 Y Y I P L I S L S 10 4593 244 A V I K I L H S K 9 4594 60 M A F D R F V A V 8 4595 81 S R I A Q I G V A 8 4596 159 S Y V L I I R T V 8 4597 239 K Q I R R A V I K 8 4598 61 A F D R F V A V S 7 4599 67 A V S N P L R Y A 7 4600 131 C T D T R I N S A 7 4601 144 A M F S T V G V D 7 4602 151 V D L L L I L L S 7 4603 161 V L I I R T V L S 7 4604 171 A S P E E R K E T 7 4605 231 P V I Y S V K T K 7 4606 10 A T D L G L S I S 6 4607 15 L S I S T L V T M 6 4608 24 L S I F W F N V R 6 4609 33 E I S F N A C L S 6 4610 68 V S N P L R Y A M 6 4611 89 A S V I R G L L M 6 4612 101 M V A L L I R L S 6 4613 103 A L L I R L S Y C 6 4614 162 L I I R T V L S V 6 4615 170 V A S P E E R K E 6 4616 191 A I Y Y I P L I S 6 4617 197 L I S L S I V H R 6 4618 212 A Y V H T M I A N 6 4619 228 L M N P V I Y S V 6 4620 237 K T K Q I R R A V 6 4621 246 I K I L H S K E T 6 4622 44 F F I K F F T V M 5 4623 59 A M A F D R F V A 5 4624 72 L R Y A M I L T D 5 4625 77 I L T D S R I A Q 5 4626 122 Y H P D V M K L S 5 4627 139 A V G L T A M F S 5 4628 178 E T F S T C V S H 5 4629 196 P L I S L S I V H 5 4630 208 K Q A P A Y V H T 5 4631 219 A N T Y L L I S P 5 4632 224 L I S P L M N P V 5 4633 2 Y Y F L S M L S A 4 4634 3 Y F L S M L S A T 4 4635 6 S M L S A T D L G 4 4636 14 G L S I S T L V T 4 4637 25 S I F W F N V R E 4 4638 27 F W F N V R E I S 4 4639 29 F N V R E I S F N 4 4640 31 V R E I S F N A C 4 4641 34 I S F N A C L S H 4 4642 43 M F F I K F F T V 4 4643 47 K F F T V M E S S 4 4644 53 E S S V L L A M A 4 4645 79 T D S R I A Q I G 4 4646 82 R I A Q I G V A S 4 4647 95 L L M L T P M V A 4 4648 104 L L I R L S Y C H 4 4649 130 S C T D T R I N S 4 4650 132 T D T R I N S A V 4 4651 137 N S A V G L T A M 4 4652 158 L S Y V L I I R T 4 4653 175 E R K E T F S T C 4 4654 200 L S I V H R F G K 4 4655 201 S I V H R F G K Q 4 4656 202 T V H R F G K Q A 4 4657 209 Q A P A Y V H T M 4 4658 213 Y V H T M I A N T 4 4659 221 T Y L L I S P L M 4 4660 223 L L I S P L M N P 4 4661 227 P L M N P V I Y S 4 4662 236 V K T K Q I R R A 4 4663 243 R A V I K I L H S 4 4664 8 L S A T D L G L S 3 4665 11 T D L G L S I S T 3 4666 13 L G L S I S T L V 3 4667 17 I S T L V T M L S 3 4668 22 T M L S I F W F N 3 4669 42 H M F F I K F F T 3 4670 46 I K F F T V M E S 3 4671 51 V M E S S V L L A 3 4672 55 S V L L A M A F D 3 4673 63 D R F V A V S N P 3 4674 76 M I L T D S R I A 3 4675 91 V I R G L L M L T 3 4676 92 I R G L L M L T P 3 4677 93 R G L L M L T P M 3 4678 99 T P M V A L L I R 3 4679 109 S Y C H S Q V L H 3 4680 111 C H S Q V L H H S 3 4681 119 S Y C Y H P D V M 3 4682 127 M K L S C T D T R 3 4683 129 L S C T D T R I N 3 4684 136 I N S A V G L T A 3 4685 143 T A M F S T V G V 3 4686 157 L L S Y V L I I R 3 4687 163 I I R T V L S V A 3 4688 164 I R T V L S V A S 3 4689 181 S T C V S H I V A 3 4690 183 C V S H I V A F A 3 4691 188 V A F A I Y Y I P 3 4692 199 S L S I V H R F G 3 4693 204 H R F G K Q A P A 3 4694 218 I A N T Y L L I S 3 4695 222 Y L L I S P L M N 3 4696 229 M N P V I Y S V K 3 4697 230 N P V I Y S V K T 3 4698 235 S V K T K Q I R R 3 4699 242 R R A V I K I L H 3 4700 4 F L S M L S A T D 2 4701 30 N V R E I S F N A 2 4702 35 S F N A C L S H M 2 4703 39 C L S H M F F I K 2 4704 45 F I K F F T V M E 2 4705 56 V L L A M A F D R 2 4706 58 L A M A F D R F V 2 4707 62 F D R F V A V S N 2 4708 71 P L R Y A M I L T 2 4709 73 R Y A M I L T D S 2 4710 74 Y A M I L T D S R 2 4711 86 I G V A S V I R G 2 4712 94 G L L M L T P M V 2 4713 106 I R L S Y C H S Q 2 4714 107 R L S Y C H S Q V 2 4715 110 Y C H S Q V L H H 2 4716 113 S Q V L H H S Y C 2 4717 117 H H S Y C Y H P D 2 4718 123 H P D V M K L S C 2 4719 125 D V M K L S C T D 2 4720 133 D T R I N S A V G 2 4721 135 R I N S A V G L T 2 4722 140 V G L T A M F S T 2 4723 141 G L T A M F S T V 2 4724 153 L L L I L L S Y V 2 4725 165 R T V L S V A S P 2 4726 166 T V L S V A S P E 2 4727 195 I P L I S L S I V 2 4728 203 V H R F G K Q A P 2 4729 207 G K Q A P A Y V H 2 4730 1 M Y Y F L S M L S 1 4731 23 M L S I F W F N V 1 4732 65 F V A V S N P L R 1 4733 80 D S R I A Q I G V 1 4734 83 I A Q I G V A S V 1 4735 85 Q I G V A S V I R 1 4736 105 L I R L S Y C H S 1 4737 120 Y C Y H P D V M K 1 4738 124 P D V M K L S C T 1 4739 126 V M K L S C T D T 1 4740 142 L T A M F S T V G 1 4741 167 V L S V A S P E E 1 4742 168 L S V A S P E E R 1 4743 169 S V A S P E E R K 1 4744 176 R K E T F S T C V 1 4745 180 F S T C V S H I V 1 4746 206 F G K Q A P A Y V 1 4747 211 P A Y V H T M I A 1 4748 232 V I Y S V K T K Q 1 4749 245 V I K T L H S K E 1 4750 HLA-B*5101 9-mers v.1: 238P1B2 195 I P L I S L S I V 24 4751 60 M A F D R F V A V 23 4752 190 F A I Y Y I P L I 23 4753 210 A P A Y V H T M I 23 4754 9 S A T D L G L S I 22 4755 58 L A M A F D R F V 22 4756 83 T A Q I G V A S V 22 4757 70 N P L R Y A M I L 21 4758 143 T A M F S T V G V 21 4759 13 L G L S I S T L V 19 4760 156 I L L S Y V L I I 18 4761 225 I S P L M N P V I 18 4762 84 A Q I G V A S V I 17 4763 88 V A S V I R G L L 17 4764 98 L T P M V A L L I 17 4765 209 Q A P A Y V H T M 17 4766 26 I F W F N V R E I 16 4767 149 V G V D L L L I L 16 4768 155 L I L L S Y V L I 16 4769 206 F G K Q A P A Y V 16 4770 218 I A N T Y L L I S 16 4771 108 L S Y C H S Q V L 15 4772 179 T F S T C V S H I 15 4773 230 N P V I Y S V K T 15 4774 240 Q I R R A V I K I 15 4775 12 D L G L S I S T L 14 4776 18 S T L V T M L S I 14 4777 43 M F F I K F F T V 14 4778 66 V A V S N P L R Y 14 4779 86 I G V A S V I R G 14 4780 102 V A L L I R L S Y 14 4781 160 Y V L I I R T V L 14 4782 162 L I I R T V L S V 14 4783 170 V A S P E E R K E 14 4784 172 S P E E R K E T F 14 4785 184 V S H I V A F A I 14 4786 192 I Y Y I P L I S L 14 4787 194 Y I P L I S L S I 14 4788 211 P A Y V H T M I A 14 4789 226 S P L M N P V I Y 14 4790 233 I Y S V K T K Q I 14 4791 238 T K Q I R R A V I 14 4792 243 R A V I K I L H S 14 4793 78 L T D S R I A Q I 13 4794 80 D S R I A Q I G V 13 4795 99 T P M V A L L I R 13 4796 118 H S Y C Y H P D V 13 4797 128 K L S C T D T R I 13 4798 141 G L T A M F S T V 13 4799 148 T V G V D L L L I 13 4800 153 L L L I L L S Y V 13 4801 187 I V A F A I Y Y I 13 4802 188 V A F A I Y Y I P 13 4803 217 M I A N T Y L L I 13 4804 228 L M N P V I Y S V 13 4805 241 I R R A V I K I L 13 4806 37 N A C L S H M F F 12 4807 38 A C L S H M F F I 12 4808 72 L R Y A M I L T D 12 4809 74 Y A M I L T D S R 12 4810 75 A M I L T D S R I 12 4811 96 L M L T P M V A L 12 4812 123 H P D V M K L S C 12 4813 159 S Y V L I I R T V 12 4814 220 N T Y L L I S P L 12 4815 5 L S M L S A T D L 11 4816 50 T V M E S S V L L 11 4817 69 S N P L R Y A M I 11 4818 93 R G L L M L T P M 11 4819 138 S A V G L T A M F 11 4820 145 M F S T V G V D L 11 4821 154 L L I L L S Y V L 11 4822 180 F S T C V S H I V 11 4823 224 L I S P L M N P V 11 4824 7 M L S A T D L G L 10 4825 16 S I S T L V T M L 10 4826 48 F F T V M E S S V 10 4827 49 F T V M E S S V L 10 4828 64 R F V A V S N P L 10 4829 87 G V A S V I R G L 10 4830 94 G L L M L T P M V 10 4831 97 M L T P M V A L L 10 4832 100 P M V A L L I R L 10 4833 121 C Y H P D V M K L 10 4834 132 T D T R I N S A V 10 4835 134 T R I N S A V G L 10 4836 140 V G L T A M F S T 10 4837 147 S T V G V D L L L 10 4838 176 R K E T F S T C V 10 4839 232 V I Y S V K T K Q 10 4840 23 M L S I F W F N V 9 4841 146 F S T V G V D L L 9 4842 158 L S Y V L I I R T 9 4843 163 I I R T V L S V A 9 4844 237 K T K Q I R R A V 9 4845 90 S V I R G L L M L 8 4846 107 R L S Y C H S Q V 8 4847 136 I N S A V G L T A 8 4848 150 G V D L L L I L L 8 4849 152 D L L L I L L S Y 8 4850 189 A F A I Y Y I P L 8 4851 215 H T M I A N T Y L 8 4852 216 T M I A N T Y L L 8 4853 15 L S I S T L V T M 7 4854 24 L S I F W F N V R 7 4855 32 R E I S F N A C L 7 4856 44 F F I K F F T V M 7 4857 63 D R F V A V S N P 7 4858 95 L L M L T P M V A 7 4859 120 Y C Y H P D V M K 7 4860 122 Y H P D V M K L S 7 4861 125 D V M K L S C T D 7 4862 133 D T R I N S A V G 7 4863 144 A M P S T V G V D 7 4864 198 I S L S I V H R F 7 4865 222 Y L L I S P L M N 7 4866 3 Y F L S M L S A T 6 4867 40 L S H M F F I K F 6 4868 46 I K F F T V M E S 6 4869 56 V L L A M A F D R 6 4870 110 Y C H S Q V L H H 6 4871 142 L T A M F S T V G 6 4872 157 L L S Y V L I I R 6 4873 166 T V L S V A S P E 6 4874 175 E R K E T F S T C 6 4875 191 A I Y Y I P L I S 6 4876 197 L I S L S I V H R 6 4877 231 P V I Y S V K T K 6 4878 1 M Y Y F L S M L S 5 4879 2 Y Y F L S M L S A 5 4880 11 T D L G L S I S T 5 4881 14 G L S I S T L V T 5 4882 51 V M E S S V L L A 5 4883 52 M E S S V L L A M 5 4884 61 A F D R F V A V S 5 4885 62 F D R F V A V S N 5 4886 76 M I L T D S R I A 5 4887 77 I L T D S R I A Q 5 4888 81 S R I A Q I G V A 5 4889 92 I R G L L M L T P 5 4890 106 I R L S Y C H S Q 5 4891 127 M K L S C T D T R 5 4892 151 V D L L L I L L S 5 4893 168 L S V A S P E E R 5 4894 181 S T C V S H I V A 5 4895 207 G K Q A P A Y V H 5 4896 213 Y V H T M I A N T 5 4897 214 V H T M I A N T Y 5 4898 229 M N P V I Y S V K 5 4899 236 V K T K Q I R R A 5 4900 8 L S A T D L G L S 4 4901 17 I S T L V T M L S 4 4902 19 T L V T M L S I F 4 4903 22 T M L S I F W F N 4 4904 25 S I F W F N V R E 4 4905 27 F W F N V R E I S 4 4906 31 V R E I S F N A C 4 4907 34 I S F N A C L S H 4 4908 47 K F F T V M E S S 4 4909 57 L L A M A F D R F 4 4910 105 L I R L S Y C H S 4 4911 111 C H S Q V L H H S 4 4912 116 L H H S Y C Y H P 4 4913 119 S Y C Y H P D V M 4 4914 129 L S C T D T R I N 4 4915 171 A S P E E R K E T 4 4916 177 K E T F S T C V S 4 4917 196 P L I S L S I V H 4 4918 234 Y S V K T K Q I R 4 4919 235 S V K T K Q I R R 4 4920 4 F L S M L S A T D 3 4921 20 L V T M L S T F W 3 4922 21 V T M L S I F W F 3 4923 30 N V R E I S F N A 3 4924 35 S F N A C L S H M 3 4925 59 A M A F D R F V A 3 4926 65 F V A V S N P L R 3 4927 91 V I R G L L M L T 3 4928 101 M V A L L T R L S 3 4929 103 A L L I R L S Y C 3 4930 126 V M K L S C T D T 3 4931 130 S C T D T R I N S 3 4932 131 C T D T R I N S A 3 4933 137 N S A V G L T A M 3 4934 164 I R T V L S V A S 3 4935 165 R T V L S V A S P 3 4936 178 E T F S T C V S H 3 4937 182 T C V S H I V A F 3 4938 186 H I V A F A I Y Y 3 4939 193 Y Y I P L I S L S 3 4940 199 S L S I V H R F G 3 4941 201 S I V H R F G K Q 3 4942 202 I V H R F G K Q A 3 4943 204 H R F G K Q A P A 3 4944 221 T Y L L I S P L M 3 4945 223 L L I S P L M N P 3 4946 227 P L M N P V I Y S 3 4947 245 V I K I L H S K E 3 4948 246 I K I L H S K E T 3 4949 6 S M L S A T D L G 2 4950 29 F N V R E I S F N 2 4951 39 C L S H M F F I K 2 4952 41 S H M F F I K F F 2 4953 45 F I K F F T V M E 2 4954 55 S V L L A M A F D 2 4955 67 A V S N P L R Y A 2 4956 73 R Y A M I L T D S 2 4957 79 T D S R I A Q I G 2 4958 104 L L I R L S Y C H 2 4959 109 S Y C H S Q V L H 2 4960 114 Q V L H H S Y C Y 2 4961 115 V L H H S Y C Y H 2 4962 124 P D V M K L S C T 2 4963 135 R I N S A V G L T 2 4964 139 A V G L T A M F S 2 4965 161 V L I I R T V L S 2 4966 173 P E E R K E T F S 2 4967 183 C V S H I V A F A 2 4968 200 L S I V H R F G K 2 4969 205 R F G K Q A P A Y 2 4970 208 K Q A P A Y V H T 2 4971 212 A Y V H T M I A N 2 4972 219 A N T Y L L I S P 2 4973 239 K Q I R R A V I K 2 4974 244 A V I K I L H S K 2 4975 10 A T D L G L S I S 1 4976 28 W F N V R E I S F 1 4977 33 E I S F N A C L S 1 4978 36 F N A C L S H M F 1 4979 42 H M F F I K F F T 1 4980 53 E S S V L L A M A 1 4981 54 S S V L L A M A F 1 4982 68 V S N P L R Y A M 1 4983 71 P L R Y A M T L T 1 4984

TABLE XIXA part 2 Pos 1 2 3 4 5 6 7 8 9 score SEQ ID HLA-A*0201 9-mers v.1B: 238P1B2 16 F L L T G V P G L 31 4985 13 S I I F L L T G V 27 4986 42 A L L G N S L I L 27 4987 32 S I P F C F L S V 22 4988 40 V T A L L G N S L 21 4989 1 F I T S T L Q N I 20 4990 9 I T S T S I I F L 20 4991 62 P M Y Y F L S M L 20 4992 23 G L E A F H T W I 19 4993 30 W I S I P F C F L 19 4994 47 S L I L F A T I T 19 4995 4 S T L Q N I T S T 18 4996 35 F C F L S V T A L 17 4997 49 I L F A T I T Q P 17 4998 51 F A T I T Q P S L 17 4999 36 C F L S V T A L L 16 5000 41 T A L L G N S L I 16 5001 43 L L G N S L I L F 16 5002 58 S L H E P M Y Y F 16 5003 46 N S L I L F A T I 15 5004 48 L I L F A T I T Q 15 5005 54 I T Q P S L H E P 15 5006 5 T L Q N I T S T S 14 5007 14 I I F L L T G V P 14 5008 18 L T G V P G L E A 14 5009 59 L H E P M Y Y F L 14 5010 6 L Q N I T S T S I 13 5011 10 T S T S I I F L L 13 5012 17 L L T G V P G L E 13 5013 44 L G N S L I L F A 13 5014 2 I T S T L Q N I T 12 5015 7 Q N I T S T S I I 12 5016 25 E A F H T W I S I 12 5017 33 I P F C F L S V T 12 5018 37 F L S V T A L L G 12 5019 11 S T S I I F L L T 11 5020 12 T S I I F L L T G 10 5021 20 G V P G L E A F H 10 5022 28 H T W I S I P F C 10 5023 39 S V T A L L G N S 10 5024 45 G N S L I L F A T 10 5025 19 T G V P G L E A F 9 5026 53 T I T Q P S L H E 9 5027 38 L S V T A L L G N 8 5028 52 A T I T Q P S L H 8 5029 8 N I T S T S I I F 7 5030 31 I S T P F C F L S 7 5031 15 I F L L T G V P G 6 5032 26 A F H T W I S I P 6 5033 50 L F A T I T Q P S 6 5034 21 V P G L E A F H T 5 5035 24 L E A F H T W I S 5 5036 34 P F C F L S V T A 5 5037 55 T Q P S L H E P M 5 5038 61 E P M Y Y F L S M 5 5039 22 P G L E A F H T W 4 5040 29 T W I S I P F C F 2 5041 3 T S T L Q N I T S 1 5042 56 Q P S L H E P M Y 1 5043 60 H E P M Y Y F L S 1 5044 57 P S L H E P M Y Y −1 5045 HLA-A1 9-mers v.1B: 238P1B2 11 S T S I I F L L T 19 5046 57 P S L H E P M Y Y 19 5047 56 Q P S L H E P M Y 16 5048 18 L T G V P G L E A 14 5049 59 L H E P M Y Y F L 14 5050 3 T S T L Q N I T S 11 5051 23 G L E A F H T W I 11 5052 32 S I P F C F L S V 11 5053 52 A T I T Q P S L H 11 5054 9 I T S T S I I F L 10 5055 12 T S I I F L L T G 10 5056 31 I S I P F C F L S 10 5057 37 F L S V T A L L G 10 5058 38 L S V T A L L G N 10 5059 54 I T Q P S L H E P 10 5060 61 E P M Y Y F L S M 10 5061 4 A L L G N S L I L 9 5062 4 S T L Q N I T S T 8 5063 40 V T A L L G N S L 8 5064 43 L L G N S L I L F 7 5065 46 N S L I L F A T I 7 5066 53 T I T Q P S L H E 7 5067 2 I T S T L Q N I T 6 5068 10 T S T S I I F L L 6 5069 17 L L T G V P G L E 6 5070 19 T G V P G L E A F 6 5071 28 H T W I S I P F C 6 5072 44 L G N S L I L F A 6 5073 16 F L L T G V P G L 5 5074 27 F H T W I S I P F 5 5075 41 T A L L G N S L I 5 5076 8 N I T S T S I I F 4 5077 25 E A F H T W I S I 4 5078 30 W I S I P F C F L 4 5079 36 C F L S V T A L L 4 5080 48 L I L F A T I T Q 4 5081 58 S L H E P M Y Y F 4 5082 22 P G L E A F H T W 3 5083 47 S L I L F A T I T 3 5084 60 H E P M Y Y F L S 3 5085 13 S I I F L L T G V 2 5086 35 F C F L S V T A L 2 5087 39 S V T A L L G N S 2 5088 1 F I T S T L Q N I 1 5089 5 T L Q N T T S T S 1 5090 14 I I F L L T G V P 1 5091 15 I F L L T G V P G 1 5092 20 G V P G L E A F H 1 5093 21 V P G L E A F H T 1 5094 26 A F H T W I S I P 1 5095 33 I P F C F L S V T 1 5096 49 I L F A T I T Q P 1 5097 51 F A T I T Q P S L 1 5098 62 P M Y Y F L S M L 1 5099 HLA-A26 9-mers v.1B: 238P1B2 58 S L H E P M Y Y F 24 5100 9 I T S T S I I F L 23 5101 43 L L G N S L I L F 23 5102 16 F L L T G V P G L 22 5103 30 W I S I P F C F L 22 5104 8 N I T S T S I I F 21 5105 40 V T A L L G N S L 21 5106 61 E P M Y Y F L S M 19 5107 13 S I I F L L T G V 18 5108 42 A L L G N S L I L 18 5109 54 I T Q P S L H E P 18 5110 1 F I T S T L Q N I 17 5111 19 T G V P G L E A F 17 5112 4 S T L Q N I T S T 16 5113 28 H T W I S I P E G 16 5114 32 S I P F C F L S V 16 5115 35 F C F L S V T A L 16 5116 36 C F L S V T A L L 16 5117 39 S V T A L L G N S 16 5118 20 G V P G L E A F H 15 5119 49 I L F A T I T Q P 15 5120 11 S T S I I F L L T 14 5121 52 A T I T Q P S L H 14 5122 59 L H E P M Y Y F L 14 5123 62 P M Y Y F L S M L 14 5124 10 T S T S I I F L L 13 5125 14 I I F L L T G V P 13 5126 29 T W I S I P F C F 13 5127 53 T I T Q P S L H E 12 5128 56 Q P S L H E P M Y 12 5129 2 I T S T L Q N I T 11 5130 18 L T G V P G L E A 11 5131 26 A F H T W I S I P 11 5132 55 T Q P S L H E P M 11 5133 57 P S L H E P M Y Y 11 5134 17 L L T G V P G L E 10 5135 27 F H T W I S I P F 10 5136 47 S L I L F A T I T 10 5137 48 L I L F A T I T Q 10 5138 51 F A T I T Q P S L 10 5139 5 T L Q N I T S T S 9 5140 25 E A F H T W I S I 9 5141 23 G L E A F H T W I 8 5142 34 P F C F L S V T A 8 5143 37 F L S V T A L L G 8 5144 50 L F A T I T Q P S 8 5145 15 I F L L T G V P G 7 5146 33 I P F C F L S V T 7 5147 12 T S I I F L L T G 6 5148 22 P G L E A F H T W 6 5149 44 L G N S L I L F A 6 5150 46 N S L I L F A T I 6 5151 38 L S V T A L L G N 5 5152 45 G N S L I L F A T 5 5153 60 H E P M Y Y F L S 5 5154 7 Q N I T S T S I I 4 5155 31 I S I P F C F L S 4 5156 3 T S T L Q N I T S 1 5157 6 L Q N I T S T S I 1 5i58 21 V P G L E A F H T 1 5159 24 L E A F H T W I S 1 5160 41 T A L L G N S L I 1 5161 HLA-A3 9-mers v.1B: 238P1B2 42 A L L G N S L I L 20 5162 20 G V P G L E A F H 19 5163 47 S L I L F A T I T 19 5164 5 T L Q N I T S T S 18 5165 49 I L F A T I T Q P 18 5166 14 I I F L L T G V P 16 5167 37 F L S V T A L L G 16 5168 58 S L H E P M Y Y F 16 5169 17 L L T G V P G L E 15 5170 43 L L G N S L I L F 15 5171 16 F L L T G V P G L 14 5172 23 G L E A F H T W I 14 5173 32 S I P F C F L S V 14 5174 39 S V T A L L G N S 14 5175 48 L I L F A T I T Q 14 5176 52 A T I T Q P S L H 14 5177 13 S I I F L L T G V 13 5178 8 N I T S T S I I F 12 5179 15 I F L L T G V P G 12 5180 12 T S I I F L L T G 11 5181 53 T I T Q P S L H E 11 5182 19 T G V P G L E A F 10 5183 46 N S L I L F A T I 10 5184 56 Q P S L H E P M Y 10 5185 57 P S L H E P M Y Y 10 5186 30 W I S I P F C F L 9 5187 33 I P F C F L S V T 9 5188 1 F I T S T L Q N I 8 5189 7 Q N I T S T S I I 8 5190 29 T W I S I P F C F 8 5191 31 I S I P F C F L S 8 5192 34 P F C F L S V T A 8 5193 41 T A L L G N S L I 8 5194 62 P M Y Y F L S M L 8 5195 22 P G L E A F H T W 7 5196 40 V T A L L G N S L 7 5197 4 S T L Q N I T S T 6 5198 18 L T G V P G L E A 6 5199 26 A F H T W I S I P 6 5200 36 C F L S V T A L L 6 5201 3 T S T L Q N I T S 5 5202 11 S T S I I F L L T 5 5203 54 I T Q P S L H E P 5 5204 61 E P M Y Y F L S M 5 5205 6 L Q N I T S T S I 4 5206 21 V P G L E A F H T 4 5207 27 F H T W I S I P F 4 5208 35 F C F L S V T A L 3 5209 38 L S V T A L L G N 3 5210 44 L G N S L I L F A 3 5211 51 F A T I T Q P S L 3 5212 59 L H E P M Y Y F L 3 5213 2 I T S T L Q N I T 2 5214 9 I T S T S I I F L 2 5215 25 E A F H T W I S I 2 5216 45 G N S L I L F A T 2 5217 2 L E A F H T W I S 1 5218 28 H T W I S I P F C 1 5219 50 L F A T I T Q P S 1 5220 HLA-B*0702 9-mers v.1B: 238P1B2 61 E P M Y Y F L S M 21 5221 33 I P F C F L S V T 18 5222 21 V P G L E A F H T 17 5223 30 W I S I P F C F L 17 5224 42 A L L G N S L I L 16 5225 9 I T S T S I I F L 14 5226 18 L T G V P G L E A 13 5227 35 F C F L S V T A L 13 5228 56 Q P S L H E P M Y 13 5229 16 F L L T G V P G L 12 5230 36 C F L S V T A L L 12 5231 40 V T A L L G N S L 12 5232 59 L H E P M Y Y F L 12 5233 11 S T S I I F L L T 11 5234 51 F A T I T Q P S L 11 5235 10 T S T S I I F L L 10 5236 62 P M Y Y F L S M L 10 5237 2 I T S T L Q N I T 9 5238 32 S I P F C F L S V 9 5239 44 L G N S L I L F A 9 5240 45 G N S L I L F A T 9 5241 58 S L H E P M Y Y F 9 5242 19 T G V P G L E A F 8 5243 23 G L E A F H T W I 8 5244 34 P F C F L S V T A 8 5245 46 N S L I L F A T I 8 5246 4 S T L Q N I T S T 7 5247 6 L Q N I T S T S I 7 5248 7 Q N I T S T S I I 7 5249 13 S I I F L L T G V 7 5250 25 E A F H T W I S I 7 5251 27 F H T W I S I P F 7 5252 43 L L G N S L I L F 7 5253 47 S L I L F A T I T 7 5254 55 T Q P S L H E P M 7 5255 1 F I T S T L Q N I 6 5256 8 N I T S T S I I F 6 5257 29 T W I S I P F C F 6 5258 41 T A L L G N S L I 6 5259 15 I F L L T G V P G 5 5260 53 T I T Q P S L H E 5 5261 20 G V P G L E A F H 4 5262 37 F L S V T A L L G 4 5263 14 I I F L L T G V P 3 5264 49 I L F A T I T Q P 3 5265 50 L F A T I T Q P S 3 5266 12 T S I I F L L T G 2 5267 17 L L T G V P G L E 2 5268 24 L E A F H T W I S 2 5269 26 A F H T W I S I P 2 5270 28 H T W I S I P F C 2 5271 31 I S I P F C F L S 2 5272 38 L S V T A L L G N 2 5273 52 A T I T Q P S L H 2 5274 54 I T Q P S L H E P 2 5275 5 T L Q N I T S T S 1 5276 22 P G L E A F H T W 1 5277 39 S V T A L L G N S 1 5278 48 L I L F A T I T Q 1 5279 HLA-B*08 9-mers v.1B: 238P1B2 16 F L L T G V P G L 18 5280 42 A L L G N S L I L 16 5281 58 S L H E P M Y Y F 16 5282 51 F A T I T Q P S L 15 5283 30 W I S I P F C F L 14 5284 35 F C F L S V T A L 14 5285 23 G L E A F H T W I 13 5286 9 I T S T S I I F L 12 5287 25 E A F H T W I S I 12 5288 40 V T A L L G N S L 12 5289 41 T A L L G N S L I 12 5290 43 L L G N S L I L F 12 5291 1 F I T S T L Q N I 11 5292 10 T S T S I I F L L 11 5293 62 P M Y Y F L S M L 11 5294 8 N I T S T S I I F 10 5295 36 C F L S V T A L L 10 5296 59 L H E P M Y Y F L 10 5297 47 S L I L F A T I T 9 5298 61 E P M Y Y F L S M 9 5299 21 V P G L E A F H T 8 5300 37 F L S V T A L L G 8 5301 49 I L F A T I T Q P 8 5302 19 T G V P G L E A F 7 5303 27 F H T W I S I P F 7 5304 33 I P F C F L S V T 7 5305 56 Q P S L H E P M Y 7 5306 5 T L Q N I T S T S 6 S307 6 L Q N I T S T S I 6 S308 7 Q N I T S T S I I 6 5309 13 S I I F L L T G V 6 5310 14 I I F L L T G V P 6 5311 17 L L T G V P G L E 6 5312 29 T W I S I P F C F 6 5313 32 S I P F C F L S V 6 5314 46 N S L I L F A T I 6 5315 48 L I L F A T I T Q 4 5316 53 T I T Q P S L H E 4 5317 4 S T L Q N I T S T 3 5318 15 I F L L T G V P G 3 5319 45 G N S L I L F A T 3 5320 3 T S T L Q N I T S 2 5321 11 S T S I I F L L T 2 5322 18 L T G V P G L E A 2 5323 22 P G L E A F H T W 2 5324 28 H T W I S I P F C 2 5325 39 S V T A L L G N S 2 5326 54 I T Q P S L H E P 2 5327 2 I T S T L Q N I T 1 5328 20 G V P G L E A F H 1 5329 26 A F H T W I S I P 1 5330 31 I S I P F C F L S 1 5331 34 P F C F L S V T A 1 5332 44 L G N S L I L F A 1 5333 60 H E P M Y Y F L S 1 5334 HLA-B*1510 9-mers v.1B: 238P1B2 59 L H E P M Y Y F L 23 5335 27 F H T W I S I P F 16 5336 9 I T S T S I I F L 14 5337 16 F L L T G V P G L 13 5338 10 T S T S I I F L L 12 5339 30 W I S I P F C F L 12 5340 35 F C F L S V T A L 12 5341 42 A L L G N S L I L 12 5342 19 T G V P G L E A F 11 5343 40 V T A L L G N S L 11 5344 51 F A T I T Q P S L 11 5345 36 C F L S V T A L L 10 5346 62 P M Y Y F L S M L 10 5347 58 S L H E P M Y Y F 9 5348 29 T W I S I P F C F 8 5349 55 T Q P S L H E P M 7 5350 61 E P M Y Y F L S M 7 5351 8 N I T S T S I I F 6 5352 43 L L G N S L I L F 6 5353 54 I T Q P S L H E P 5 5354 14 I I F L L T G V P 4 5355 15 I F L L T G V P G 4 5356 31 I S I P F C F L S 4 5357 33 I P F C F L S V T 4 5358 2 I T S T L Q N I T 3 5359 4 S T L Q N I T S T 3 5360 18 L T G V P G L E A 3 5361 34 P F C F L S V T A 3 5362 45 G N S L I L F A T 3 5363 49 I L F A T I T Q P 3 5364 53 T I T Q P S L H E 3 5365 3 T S T L Q N I T S 2 5366 5 T L Q N T T S T S 2 5367 12 T S I I F L L T G 2 5368 17 L L T G V P G L E 2 5369 20 G V P G L E A F H 2 5370 22 P G L E A F H T W 2 5371 23 G L E A F H T W I 2 5372 28 H T W I S I P F C 2 5373 37 F L S V T A L L G 2 5374 41 T A L L G N S L I 2 5375 56 Q P S L H E P M Y 2 5376 11 S T S I I F L L T 1 5377 24 L E A F H T W I S 1 5378 25 E A F H T W I S I 1 5379 26 A F H T W I S I P 1 5380 44 L G N S L I L F A 1 5381 46 N S L I L F A T I 1 5382 48 L I L F A T I T Q 1 5383 50 L F A T I T Q P S 1 5384 52 A T I T Q P S L H 1 5385 57 P S L H E P M Y Y 1 5386 HLA-B*2705 9-mers v.1B: 238P1B2 35 F C F L S V T A L 18 5387 42 A L L G N S L I L 17 5388 9 I T S T S I I F L 16 5389 16 F L L T G V P G L 16 5390 19 T G V P G L E A F 15 5391 20 G V P G L E A F H 15 5392 10 T S T S I T F L L 14 5393 36 C F L S V T A L L 14 5394 40 V T A L L G N S L 14 5395 51 F A T I T Q P S L 14 5396 62 P M Y Y F L S M L 14 5397 27 F H T W I S I P F 13 5398 29 T W I S I P F C F 13 5399 41 T A L L G N S L I 13 5400 43 L L G N S L I L F 13 5401 58 S L H E P M Y Y F 13 5402 8 N I T S T S I I F 12 5403 52 A T I T Q P S L H 12 5404 57 P S L H E P M Y Y 12 5405 59 L H E P M Y Y F L 12 5406 23 G L E A F H T W I 11 5407 25 E A F H T W I S I 11 5408 30 W I S I P F C F L 11 5409 46 N S L I L F A T I 11 5410 4 S T L Q N I T S T 10 5411 6 L Q N I T S T S I 10 5412 7 Q N I T S T S T I 10 5413 49 I L F A T I T Q P 10 5414 56 Q P S L H E P M Y 10 5415 61 E P M Y Y F L S M 10 5416 1 F I T S T L Q N I 9 5417 55 T Q P S L H E P M 9 5418 14 I I F L L T G V P 7 5419 33 I P F G F L S V T 7 5420 13 S I I F L L T G V 6 5421 45 G N S L I L F A T 6 5422 47 S L I L F A T I T 6 5423 2 I T S T L Q N I T 5 5424 12 T S I I F L L T G 5 5425 15 I F L L T G V P G 5 5426 26 A F H T W I S I P 5 5427 28 H T W I S I P F C 5 5428 44 L G N S L I L F A 5 5429 53 T I T Q P S L H E 5 5430 54 I T Q P S L H E P 5 5431 3 T S T L Q N I T S 4 5432 22 P G L E A F H T W 4 5433 31 I S I P F C F L S 4 5434 34 P F C F L S V T A 4 5435 39 S V T A L L G N S 4 5436 48 L I L F A T I T Q 4 5437 5 T L Q N I T S T S 3 5438 17 L L T G V P G L E 3 5439 18 L T G V P G L E A 3 5440 21 V P G L E A F H T 3 5441 38 L S V T A L L G N 3 5442 11 S T S I I F L L T 2 5443 32 S I P F C F L S V 2 5444 37 F L S V T A L L G 2 5445 24 L E A F H T W I S 1 5446 50 L F A T I T Q P S 1 5447 HLA-B*2709 9-mers v.1B: 238P1B2 16 F L L T G V P G L 14 5448 42 A L L G N S L I L 14 5449 10 T S T S I I F L L 13 5450 35 F C F L S V T A L 13 5451 36 C F L S V T A L L 13 5452 51 F A T I T Q P S L 12 5453 59 L H E P M Y Y F L 12 5454 62 P M Y Y F L S M L 12 5455 1 F I T S T L Q N I 11 5456 9 I T S T S I I F L 11 5457 23 G L E A F H T W I 11 5458 29 T W I S I P F C F 11 5459 8 N I T S T S I I F 10 5460 19 T G V P G L E A F 10 5461 25 E A F H T W I S I 10 5462 27 F H T W I S I P F 10 5463 30 W I S I P F C F L 10 5464 40 V T A L L G N S L 10 5465 41 T A L L G N S L I 10 5466 46 N S L I L F A T I 10 5467 7 Q N I T S T S I I 9 5468 13 S I I F L L T G V 9 5469 32 S I P F C F L S V 9 5470 55 T Q P S L H E P M 9 5471 58 S L H E P M Y Y F 9 5472 61 E P M Y Y F L S M 9 5473 6 L Q N I T S T S I 8 5474 43 L L G N S L I L F 8 5475 15 I F L L T G V P G 4 5476 45 G N S L I L F A T 4 5477 49 I L F A T I T Q P 4 5478 14 I I F L L T G V P 3 5479 20 G V P G L E A F H 3 5480 31 I S I P F C F L S 3 5481 33 I P F C F L S V T 3 5482 4 S T L Q N I T S T 2 5483 11 S T S I I F L L T 2 5484 12 T S I I F L L T G 2 5485 22 P G L E A F H T W 2 5486 37 F L S V T A L L G 2 5487 38 L S V T A L L G N 2 5488 39 S V T A L L G N S 2 5489 44 L G N S L I L F A 2 5490 47 S L I L F A T I T 2 5491 48 L I L F A T I T Q 2 5492 52 A T I T Q P S L H 2 5493 53 T I T Q P S L H E 2 5494 54 I T Q P S L H E P 2 5495 57 P S L H E P M Y Y 2 5496 2 I T S T L Q N I T 1 5497 3 T S T L Q N I T S 1 5498 5 T L Q N I T S T S 1 5499 17 L L T G V P G L E 1 5500 18 L T G V P G L E A 1 5501 21 V P G L E A F H T 1 5502 26 A F H T W I S I P 1 5503 28 H T W I S I P F C 1 5504 34 P F C F L S V T A 1 5505 50 L F A T I T Q P S 1 5506 56 Q P S L H E P M Y 1 5507 HLA-B*4402 9-mers v.1B: 238P1B2 42 A L L G N S L I L 17 5508 9 I T S T S I I F L 16 5509 19 T G V P G L E A F 16 5510 35 F C F L S V T A L 16 5511 10 T S T S I I F L L 15 5512 29 T W I S I P F C F 15 5513 2 P G L E A F H T W 14 5514 43 L L G N S L I L F 14 5515 7 Q N I T S T S I I 13 5S16 16 F L L T G V P G L 13 5517 25 E A F H T W I S I 13 5518 36 C F L S V T A L L 13 5519 59 L H E P M Y Y F L 13 5520 8 N I T S T S I I F 12 5521 27 F H T W I S I P F 12 5522 30 W I S I P F C F L 12 5523 40 V T A L L G N S L 12 5524 46 N S L I L F A T I 12 5525 56 Q P S L H E P M Y 12 5526 57 P S L H E P M Y Y 12 5527 60 H E P M Y Y F L S 12 5528 24 L E A F H T W I S 11 5529 41 T A L L G N S L I 11 5530 58 S L H E P M Y Y F 11 5531 62 P M Y Y F L S M L 11 5532 51 F A T I T Q P S L 10 5533 1 F I T S T L Q N I 9 5534 52 A T I T Q P S L H 9 5535 6 L Q N I T S T S I 8 5536 23 G L E A F H T W I 8 5537 26 A F H T W I S I P 7 5538 31 I S I P F C F L S 7 5539 4 S T L Q N I T S T 6 5540 11 S T S I I F L L T 6 5541 12 T S I I F L L T G 6 5542 45 G N S L I L F A T 6 5543 47 S L I L F A T I T 6 5544 49 I L F A T I T Q P 6 5545 13 S I I F L L T G V 5 5546 14 I I F L L T G V P 5 5547 54 I T Q P S L H E P 5 5548 61 E P M Y Y F L S M 5 5549 5 T L Q N I T S T S 4 5550 32 S I P F C F L S V 4 5551 33 I P F C F L S V T 4 5552 37 F L S V T A L L G 4 5553 44 L G N S L I L F A 4 5554 48 L I L F A T I T Q 4 5555 2 I T S T L Q N I T 3 5556 3 T S T L Q N I T S 3 5557 15 I F L L T G V P G 3 5558 17 L L T G V P G L E 3 5559 28 H T W I S I P F C 3 5560 38 L S V T A L L G N 3 5561 50 L F A T I T Q P S 3 5562 20 G V P G L E A F H 2 5563 21 V P G L E A F H T 2 5564 34 P F C F L S V T A 2 5565 39 S V T A L L G N S 2 5566 18 L T G V P G L E A 1 5567 53 T I T Q P S L H E 1 5568 55 T Q P S L H E P M 1 5569 HLA-B*5101 9-mers v.1B: 238P1B2 41 T A L L G N S L I 24 5570 25 E A F H T W I S I 23 5571 51 F A T I T Q P S L 18 5572 33 I P F C F L S V T 17 5573 46 N S L I L F A T I 16 5574 6 L Q N I T S T S I 14 5575 1 F I T S T L Q N I 13 5576 21 V P G L E A F H T 13 5577 22 P G L E A F H T W 13 5578 61 E P M Y Y F L S M 13 5579 7 Q N I T S T S I I 12 5580 9 I T S T S I I F L 12 5581 16 F L L T C V P G L 12 5582 23 G L E A F H T W I 12 5583 35 F C F L S V T A L 12 5584 32 S I P F C F L S V 11 5585 62 P M Y Y F L S M L 11 5586 13 S I I F L L T G V 10 5587 36 C F L S V T A L L 10 5588 42 A L L G N S L I L 10 5589 44 L G N S L I L F A 10 5590 56 Q P S L H E P M Y 10 5591 59 L H E P M Y Y F L 9 5592 10 T S T S I I F L L 8 5593 19 T G V P G L E A F 8 5594 40 V T A L L G N S L 8 5595 30 W I S I P F C F L 7 5596 48 L I L F A T I T Q 7 5597 49 I L F A T I T Q P 7 5598 14 I I F L L T G V P 6 5599 15 I F L L T G V P G 6 5600 17 L L T G V P G L E 6 5601 3 T S T L Q N I T S 5 5602 12 T S I I F L L T G 5 5603 38 L S V T A L L G N 5 5604 43 L L G N S L I L F 5 5605 2 I T S T L Q N I T 4 5606 4 S T L Q N I T S T 4 5607 37 F L S V T A L L G 4 5608 54 I T Q P S L H E P 4 5609 5 T L Q N I T S T S 3 5610 8 N I T S T S I I F 3 5611 18 L T G V P G L E A 3 5612 28 H T W I S T P F C 3 5613 34 P F C F L S V T A 3 5614 50 L F A T I T Q P S 3 5615 53 T I T Q P S L H E 3 5616 57 P S L H E P M Y Y 3 5617 11 S T S I I F L L T 2 5618 20 G V P G L E A F H 2 5619 24 L E A F H T W I S 5620

TABLE XIXA part 3 Pos 1 2 3 4 5 6 7 8 9 score SEQ ID HLA-A*0201 9-mers v.2: 238P1B2 8 L T S P L M N P V 20 5621 4 N T Y L L T S P L 18 5622 7 L L T S P L M N P 16 5623 1 M I A N T Y L L T 15 5624 2 I A N T Y L L T S 15 5625 6 Y L L T S P L M N 15 5626 9 T S P L M N P V I 9 5627 3 A N T Y L L T S P 7 5628 5 T Y L L T S P L M 7 5629 HLA-A1 9-mers v.2: 238P1B2 6 Y L L T S P L M N 10 5630 8 L T S P L M N P V 10 5631 1 M I A N T Y L L T 8 5632 2 I A N T Y L L T S 7 5633 4 N T Y L L T S P L 6 5634 9 T S P L M N P V I 6 5635 5 T Y L L T S P L M 2 5636 7 L L T S P L M N P 2 5637 3 A N T Y L L T S P 1 5638 HLA-A26 9-mers v.2: 238P1B2 4 N T Y L L T S P L 20 5639 8 L T S P L M N P V 17 5640 1 M I A N T Y L L T 15 5641 7 L L T S P L M N P 15 5642 6 Y L L T S P L M N 10 5643 5 T Y L L T S P L M 9 5644 3 A N T Y L L T S P 6 5645 2 T A N T Y L L T S 4 5646 9 T S P L M N P V I 2 5647 HLA-A3 9-mers v.2: 238P1B2 6 Y L L T S P L M N 17 5648 1 M I A N T Y L L T 13 5649 7 L L T S P L M N P 11 5650 2 I A N T Y L L T S 9 5651 4 N T Y L L T S P L 8 5652 3 A N T Y L L T S P 6 5653 9 T S P L M N P V I 5 5654 5 T Y L L T S P L M 3 5655 8 L T S P L M N P V 3 5656 HLA-B*0702 9-mers v.2: 238P1B2 4 N T Y L L T S P L 12 5657 8 L T S P L M N P V 10 5658 1 M I A N T Y L L T 9 5659 9 T S P L M N P V I 8 S660 5 T Y L L T S P L M 7 5661 2 I A N T Y L L T S 3 5662 3 A N T Y L L T S P 3 5663 7 L L T S P L M N P 3 5664 6 Y L L T S P L M N 2 5665 HLA-B*08 9-mers v.2 238P1B2 4 N T Y L L T S P L 11 5666 9 T S P L M N P V I 8 5667 6 Y L L T S P L M N 6 5668 7 L L T S P L M N P 6 5669 2 I A N T Y L L T S 5 5670 1 M I A N T Y L L T 4 5671 3 A N T Y L L T S P 1 5672 5 T Y L L T S P L M 1 5673 HLA-B*1510 9-mers v.2 238P1B2 4 N T Y L L T S P L 10 5674 5 T Y L L T S P L M 8 5675 8 L T S P L M N P V 4 5676 9 T S P L M N P V I 4 5677 2 I A N T Y L L T S 3 5678 6 Y L L T S P L M N 2 5679 1 M I A N T Y L L T 1 5680 7 L L T S P L M N P 1 5681 HLA-B*2705 9-mers v.2 238P1B2 4 N T Y L L T S P L 15 5682 5 T Y L L T S P L M 13 5683 9 T S P L M N P V I 10 5684 3 A N T Y L L T S P 6 5685 7 L L T S P L M N P 5 5686 2 I A N T Y L L T S 4 5687 6 Y L L T S P L M N 4 5688 8 L T S P L M N P V 3 5689 1 M I A N T Y L L T 1 5690 HLA-B*2709 9-mers v.2 238P1B2 4 N T Y L L T S P L 12 5691 5 T Y L L T S P L M 11 5692 8 L T S P L M N P V 9 5693 9 T S P L M N P V I 9 5694 6 Y L L T S P L M N 3 5695 7 L L T S P L M N P 3 5696 2 I A N T Y L L T S 2 5697 3 A N T Y L L T S P 2 5698 1 M I A N T Y L L T 1 5699 HLA-B*4402 9-mers v.2 238P1B2 4 N T Y L L T S P L 13 5700 9 T S P L M N P V I 11 5701 8 L T S P L M N P V 5702

TABLE XIXB part 1 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID HLA-A*0201 10-mers v.1: 238P1B2 161 V L I I R T V L S V 30 5703 8 R I A Q I G V A S V 28 5704 9 L M L T P M V A L L 28 5705 7 I L T D S R I A Q I 26 5706 95 L L M L T P M V A L 26 5707 191 A I Y Y I P L I S L 26 5708 25 S I F W F N V R E I 25 5709 152 D L L L I L L S Y V 25 5710 153 L L L I L L S Y V L 25 5711 155 L I L L S Y V L I I 25 5712 223 L L I S P L M N P V 25 5713 227 P L M N P V I Y S V 25 5714 6 S M L S A T D L G L 24 5715 5 L L A M A F D R F V 24 5716 5 A M A F D R F V A V 24 5717 154 L L I L L S Y V L I 24 5718 19 Y I P L I S L S I V 24 5719 1 G L S I S T L V T M 23 5720 14 A M F S T V G V D L 23 5721 4 F L S M L S A T D L 21 5722 142 L T A M F S T V G V 21 5723 147 S T V G V D L L L I 21 5724 186 H I V A F A I Y Y I 21 5725 22 L I S P L M N P V I 21 5726 24 Q I R R A V I K I L 21 5727 11 T D L G L S I S T L 20 5728 22 T M L S I F W F N V 20 5729 42 H M F F I K F F T V 20 5730 97 M L T P M V A L L I 20 5731 162 L I I R T V L S V A 20 5732 12 D L G L S I S T L V 19 5733 107 R L S Y C H S Q V L 19 5734 12 Y C Y H P D V M K L 19 5735 148 T V G V D L L L I L 19 5736 157 L L S Y V L I I R T 19 5737 158 L S Y V L I I R T V 19 5738 21 T M I A N T Y L L I 19 5739 15 L S I S T L V T M L 18 5740 51 V M E S S V L L A M 18 5741 156 I L L S Y V L I I R 18 5742 22 Y L L I S P L M N P 18 5743 49 F T V M E S S V L L 17 5744 8 I G V A S V I R G L 17 5745 87 G V A S V I R G L L 17 5746 89 A S V I R G L L M L 17 5747 94 G L L M L T P M V A 17 5748 189 A F A I Y Y I P L I 17 5749 193 Y Y I P L I S L S I 17 5750 232 V I Y S V K T K Q I 17 5751 239 K Q I R R A V I K I 17 5752 50 T V M E S S V L L A 16 5753 75 A M I L T D S R I A 16 5754 90 S V I R G L L M L T 16 5755 133 D T R I N S A V G L 16 5756 135 R I N S A V G L T A 16 5757 149 V G V D L L L I L L 16 5758 188 V A F A I Y Y I P L 16 5759 208 K Q A P A Y V H T M 16 5760 215 H T M I A N T Y L L 16 5761 8 L S A T D L G L S I 15 5762 74 Y A M I L T D S R I 15 5763 99 T P M V A L L I R L 15 5764 103 A L L I R L S Y C H 15 5765 131 C T D T R I N S A V 15 5766 140 V G L T A M F S T V 15 5767 196 P L I S L S I V H R 15 5768 197 L I S L S I V H R F 15 5769 217 M I A N T Y L L I S 15 5770 7 M L S A T D L G L S 14 5771 47 K F F T V M E S S V 14 5772 68 V S N P L R Y A M I 14 5773 83 I A Q I G V A S V I 14 5774 91 V I R G L L M L T P 14 5775 104 L L I R L S Y C H S 14 5776 106 I R L S Y C H S Q V 14 5777 145 M F S T V G V D L L 14 5778 178 E T F S T C V S H I 14 5779 219 A N T Y L L I S P L 14 5780 237 K T K Q I R R A V I 14 5781 45 F I K F F T V M E S 13 5782 56 V L L A M A F D R F 13 5783 66 V A V S N P L R Y A 13 5784 71 P L R Y A M I L T D 13 5785 93 R G L L M L T P M V 13 5786 105 L I R L S Y C H S Q 13 5787 130 S C T D T R I N S A 13 5788 163 I I R T V L S V A S 13 5789 167 V L S V A S P E E R 13 5790 181 S T C V S H I V A F 13 5791 183 C V S H I V A F A I 13 5792 199 S L S I V H R F G K 13 5793 201 S I V H R F C K Q A 13 5794 218 I A N T Y L L I S P 13 5795 228 L M N P V I Y S V K 13 5796 235 S V K T K Q I R R A 13 5797 245 V I K I L H S K E T 13 5798 2 Y Y F L S M L S A T 12 5799 1 A T D L G L S I S T 12 5800 17 I S T L V T M L S I 12 5801 18 S T L V T M L S I F 12 5802 31 V R E I S F N A C L 12 5803 37 N A C L S H M F F I 12 5804 39 C L S H M F F I K F 12 5805 60 M A F D R F V A V S 12 5806 67 A V S N P L R Y A M 12 5807 69 S N P L R Y A M I L 12 5808 85 Q I G V A S V I R G 12 5809 115 V L H H S Y C Y H P 12 5810 139 A V G L T A M F S T 12 5811 159 S Y V L I I R T V L 12 5812 170 V A S P E E R K E T 12 5813 205 R F G K Q A P A Y V 12 5814 209 Q A P A Y V H T M I 12 5815 212 A Y V H T M I A N T 12 5816 23 M L S I F W F N V R 11 5817 34 I S F N A C L S H M 11 5818 58 L A M A F D R F V A 11 5819 63 D R F V A V S N P L 11 5820 76 M I L T D S R I A Q 11 5821 79 T D S R I A Q I G V 11 5822 98 L T P M V A L L I R 11 5823 102 V A L L I R L S Y C 11 5824 128 K L S C T D T R I N 11 5825 136 I N S A V G L T A M 11 5826 146 F S T V G V D L L L 11 5827 179 T F S T C V S H I V 11 5828 236 V K T K Q I R R A V 11 5829 243 R A V I K I L H S K 11 5830 9 S A T D L G L S I S 10 5831 16 S I S T L V T M L S 10 5832 19 T L V T M L S I F W 10 5833 48 F F T V M E S S V L 10 5834 52 M E S S V L L A M A 10 5835 88 V A S V I R G L L M 10 5836 92 I R G L L M L T P M 10 5837 117 H H S Y C Y H P D V 10 5838 126 V M K L S C T D T R 10 5839 127 M K L S C T D T R I 10 5840 134 T R I N S A V G L T 10 5841 141 G L T A M F S T V G 10 5842 150 G V D L L L I L L S 10 5843 151 V D L L L I L L S Y 10 5844 160 Y V L I I R T V L S 10 5845 169 S V A S P E E R K E 10 5846 182 T C V S H I V A F A 10 5847 214 V H T M I A N T Y L 10 5848 220 N T Y L L I S P L M 10 5849 244 A V I K I L H S K E 10 5850 13 L G L S I S T L V T 9 5851 20 L V T M L S I F W F 9 5852 100 P M V A L L I R L S 9 58S3 101 M V A L L I R L S Y 9 5854 164 I R T V L S V A S P 9 5855 166 T V L S V A S P E E 9 5856 213 Y V H T M I A N T Y 9 5857 3 Y F L S M L S A T D 8 5858 30 N V R E I S F N A C 8 5859 55 S V L L A M A F D R 8 5860 65 F V A V S N P L R Y 8 5861 84 A Q I G V A S V I R 8 5862 123 H P D V M K L S C T 8 5863 125 D V M K L S C T D T 8 5864 138 S A V G L T A M F S 8 5865 143 T A M F S T V G V D 8 5866 175 E R K E T F S T C V 8 5867 187 I V A F A I Y Y I P 8 5868 226 S P L M N P V I Y S 8 5869 229 M N P V I Y S V K T 8 5870 1 M Y Y F L S M L S A 7 5871 33 E I S F N A C L S H 7 5872 72 L R Y A M I L T D S 7 5873 80 D S R I A Q I G V A 7 5874 81 S R I A Q I G V A S 7 5875 109 S Y C H S Q V L H H 7 5876 11 Y C H S Q V L H H S 7 5877 190 F A I Y Y I P L I S 7 5878 192 I Y Y I P L I S L S 7 5879 202 I V H R F G K Q A P 7 5880 207 G K Q A P A Y V H T 7 5881 210 A P A Y V H T M I A 7 5882 242 R R A V I K I L H S 7 5883 21 V T M L S I F W F N 6 5884 41 S H M F F I K F F T 6 5885 61 A F D R F V A V S N 6 5886 70 N P L R Y A M I L T 6 5887 78 L T D S R I A Q I G 6 5888 114 Q V L H H S Y C Y H 6 5889 122 Y H P D V M K L S C 6 5890 171 A S P E E R K E T F 6 5891 200 L S I V H R F G K Q 6 5892 203 V H R F G K Q A P A 6 5893 24 L S I F W F N V R E 5 5894 28 W F N V R E I S F N 5 5895 29 F N V R E I S F N A 5 5896 40 L S H M F F I K F F 5 5897 43 M F F I K F F T V M 5 5898 44 F F I K F F T V M E 5 5899 108 L S Y C H S Q V L H 5 5900 118 H S Y C Y H P D V M 5 5901 204 H R F G K Q A P A Y 5 5902 230 N P V I Y S V K T K 5 5903 27 F W F N V R E I S F 4 5904 35 S F N A C L S H M F 4 5905 38 A C L S H M F F I K 4 5906 46 I K F F T V M E S S 4 5907 54 S S V L L A M A F D 4 5908 62 F D R F V A V S N P 4 5909 73 R Y A M I L T D S R 4 5910 137 N S A V G L T A M F 4 5911 165 R T V L S V A S P E 4 5912 180 F S T C V S H I V A 4 5913 185 S H I V A F A I Y Y 4 5914 195 I P L T S L S I V H 4 5915 198 I S L S I V H R F G 4 5916 225 I S P L M N P V I Y 4 5917 231 P V I Y S V K T K Q 4 5918 5 L S M L S A T D L G 3 5919 36 F N A C L S H M F F 3 5920 111 C H S Q V L H H S Y 3 5921 113 S Q V L H H S Y C Y 3 5922 116 L H H S Y C Y H P D 3 5923 119 S Y C Y H P D V M K 3 5924 172 S P E E R K H T F S 3 5925 177 K E T F S T C V S H 3 5926 184 V S H I V A F A I Y 3 5927 211 P A Y V H T M I A N 3 5928 221 T Y L L I S P L M N 3 5929 26 I F W F N V R E I S 2 5930 32 R E I S F N A C L S 2 5931 129 L S C T D T R I N S 2 5932 168 L S V A S P E E R K 2 5933 173 P E E R K E T F S T 2 5934 233 I Y S V K T K Q I R 2 5935 234 Y S V K T K Q I R R 2 5936 238 T K Q I R R A V I K 2 5937 241 I R R A V I K I L H 2 5938 64 R F V A V S N P L R 1 5939 121 C Y H P D V M K L S 1 5940 132 T D T R I N S A V G 1 5941 206 F G K Q A P A Y V H 1 5942 124 P D V M K L S C T D −1 5943 174 E E R K E T F S T C −1 5944 176 R K E T F S T C V S −1 5945 53 E S S V L L A M A F −2 5946 HLA-A*0202 10-mers v.1: 238P1B2 59 A M A F D R F V A V 4 5947 189 A F A I Y Y I P L I 4 5948 210 A P A Y V H T M I A 4 5949 8 L S A T D L G L S I 3 5950 36 F N A C L S H M F F 3 5951 57 L L A M A F D R F V 3 5952 65 F V A V S N P L R Y 3 5953 73 R Y A M I L T D S R 3 5954 82 R I A Q I G V A S V 3 5955 87 G V A S V I R G L L 3 5956 101 M V A L L I R L S Y 3 5957 137 N S A V G L T A M F 3 5958 142 L T A M F S T V G V 3 5959 169 S V A S P E E R K E 3 5960 187 I V A F A I Y Y I P 3 5961 208 K Q A P A Y V H T M 3 5962 217 M I A N T Y L L I S 3 5963 242 R R A V I K I L H S 3 5964 9 S A T D L G L S I S 2 5965 37 N A C L S H M F F I 2 5966 58 L A M A F D R F V A 2 5967 60 M A F D R F V A V S 2 5968 66 V A V S N P L R Y A 2 5969 74 Y A M I L T D S R I 2 5970 83 I A Q I G V A S V I 2 5971 88 V A S V I R G L L M 2 5972 102 V A L L I R L S Y C 2 5973 138 S A V G L T A M F S 2 5974 143 T A M F S T V G V D 2 5975 170 V A S P E E R K E T 2 5976 188 V A F A I Y Y I P L 2 5977 190 F A I Y Y I P L I S 2 5978 209 Q A P A Y V H T M I 2 5979 211 P A Y V H T M I A N 2 5980 218 I A N T Y L L I S P 2 5981 243 R A V I K I L H S K 2 5982 10 A T D L G L S I S T 1 5983 38 A C L S H M F F I K 1 5984 61 A F D R F V A V S N 1 5985 67 A V S N P L R Y A M 1 5986 75 A M I L T D S R I A 1 5987 84 A Q I G V A S V I R 1 5988 89 A S V I R G L L M L 1 5989 103 A L L I R L S Y C H 1 5990 139 A V G L T A M F S T 1 5991 144 A M F S T V G V D L 1 5992 171 A S P E E R K E T F 1 5993 191 A I Y Y I P L I S L 1 5994 212 A Y V H T M I A N T 1 5995 219 A N T Y L L I S P L 1 5996 244 A V I K I L H S K E 1 5997 HLA-A*0203 10-mers v.1: 238P1B2 52 M E S S V L L A M A 18 5998 182 T C V S H I V A F A 18 5999 203 V H R F G K Q A P A 18 6000 1 M Y Y F L S M L S A 10 6001 29 F N V R E I S F N A 10 6002 50 T V M E S S V L L A 10 6003 58 L A M A F D R F V A 10 6004 66 V A V S N P L R Y A 10 6005 75 A M I L T D S R I A 10 6006 80 D S R I A Q I G V A 10 6007 94 G L L M L T P M V A 10 6008 130 S C T D T R I N S A 10 6009 135 R I N S A V G L T A 10 6010 162 L I I R T V L S V A 10 6011 180 F S T C V S H I V A 10 6012 201 S I V H R F G K Q A 10 6013 210 A P A Y V H T M I A 10 6014 235 S V K T K Q I R R A 10 6015 2 Y Y F L S M L S A T 9 6016 30 N V R E I S F N A C 9 6017 51 V M E S S V L L A M 9 6018 53 E S S V L L A M A F 9 6019 59 A M A F D R F V A V 9 6020 67 A V S N P L R Y A M 9 6021 76 M I L T D S R I A Q 9 6022 81 S R I A Q I G V A S 9 6023 95 L L M L T P M V A L 9 6024 131 C T D T R I N S A V 9 6025 136 I N S A V G L T A M 9 6026 163 I I R T V L S V A S 9 6027 181 S T C V S H I V A F 9 6028 183 C V S H I V A F A I 9 6029 202 I V H R F G K Q A P 9 6030 204 H R F G K Q A P A Y 9 6031 211 P A Y V H T M I A N 9 6032 236 V K T K Q I R R A V 9 6033 3 Y F L S M L S A T D 8 6034 31 V R E I S F N A C L 8 6035 54 S S V L L A M A F D 8 6036 60 M A F D R F V A V S 8 6037 68 V S N P L R Y A M I 8 6038 77 I L T D S R I A Q I 8 6039 82 R I A Q I G V A S V 8 6040 96 L M L T P M V A L L 8 6041 132 T D T R I N S A V G 8 6042 137 N S A V G L T A M F 8 6043 164 I R T V L S V A S P 8 6044 184 V S H I V A F A I Y 8 6045 205 R F G K Q A P A Y V 8 6046 212 A Y V H T M I A N T 8 6047 237 K T K Q I R R A V I 8 6048 HLA-A1 10-mers v.1: 238P1B2 184 V S H I V A F A I Y 23 6049 65 F V A V S N P L R Y 22 6050 101 M V A L L I R L S Y 22 6051 151 V D L L L I L L S Y 22 6052 10 A T D L G L S I S T 21 6053 185 S H I V A F A I Y Y 21 6054 225 I S P L M N P V I Y 19 6055 150 G V D L L L I L L S 18 6056 51 V M E S S V L L A M 17 6057 113 S Q V L H H S Y C Y 17 6058 131 C T D T R I N S A V 17 6059 147 S T V G V D L L L I 17 6060 213 Y V H T M I A N T Y 17 6061 78 L T D S R I A Q I G 16 6062 204 H R F G K Q A P A Y 16 6063 111 C H S Q V L H H S Y 15 6064 97 M L T P M V A L L I 13 6065 146 F S T V G V D L L L 13 6066 31 V R E I S F N A C L 12 6067 89 A S V I R G L L M L 12 6068 98 L T P M V A L L I R 12 6069 172 S P E E R K E T F S 12 6070 193 Y Y I P L I S L S I 12 6071 8 L S A T D L G L S I 11 6072 61 A F D R F V A V S N 11 6073 176 R K E T F S T C V S 11 6074 17 I S T L V T M L S I 10 6075 21 V T M L S I F W F N 10 6076 39 C L S H M F F I K F 10 6077 68 V S N P L R Y A M I 10 6078 88 V A S V I R G L L M 10 6079 123 H P D V M K L S C T 10 6080 173 P E E R K H T F S T 10 6081 180 F S T C V S H I V A 10 6082 234 Y S V K T K Q I R R 10 6083 49 F T V M E S S V L L 9 6084 50 T V M E S S V L L A 9 6085 109 S Y C H S Q V L H H 9 6086 129 L S C T D T R I N S 9 6087 161 V L I I R T V L S V 9 6088 200 L S I V H R F G K Q 9 6089 6 S M L S A T D L G L 8 6090 18 S T L V T M L S I F 8 6091 91 V I R G L L M L T P 8 6092 122 Y H P D V M K L S C 8 6093 181 S T C V S H I V A F 8 6094 215 H T M I A N T Y L L 8 6095 216 T M I A N T Y L L I 8 6096 220 N T Y L L I S P L M 8 6097 237 K T K Q I R R A V I 8 6098 13 L G L S I S T L V T 7 6099 71 P L R Y A M I L T D 7 6100 135 R I N S A V G L T A 7 6101 149 V G V D L L L I L L 7 6102 155 L I L L S Y V L I I 7 6103 178 E T F S T C V S H I 7 6104 190 F A I Y Y I P L I S 7 6105 221 T Y L L I S P L M N 7 6106 226 S P L M N P V I Y S 7 6107 239 K Q I R R A V I K I 7 6108 1 M Y Y F L S M L S A 6 6109 5 L S M L S A T D L G 6 6110 15 L S I S T L V T M L 6 6111 33 E I S F N A C L S H 6 6112 54 S S V L L A M A F D 6 6113 70 N P L R Y A M I L T 6 6114 108 L S Y C H S Q V L H 6 6115 121 C Y H P D V M K L S 6 6116 133 D T R I N S A V G L 6 6117 142 L T A M F S T V G V 6 6118 148 T V G V D L L L I L 6 6119 165 R T V L S V A S P E 6 6120 170 V A S P E E R K E T 6 6121 217 M I A N T Y L L I S 6 6122 224 L I S P L M N P V I 6 6123 241 I R R A V I K I L H 6 6124 242 R R A V I K I L H S 6 6125 19 T L V T M L S I F W 5 6126 23 M L S I F W F N V R 5 6127 24 L S I F W F N V R E 5 6128 27 F W F N V R E I S F 5 6129 56 V L L A M A F D R F 5 6130 76 M I L T D S R I A Q 5 6131 80 D S R I A Q I G V A 5 6132 85 Q I G V A S V I R G 5 6133 118 H S Y C Y H P D V M 5 6134 120 Y C Y H P D V M K L 5 6135 137 N S A V G L T A M F 5 6136 156 I L L S Y V L I I R 5 6137 157 L L S Y V L I I R T 5 6138 171 A S P E E R K E T F 5 6139 188 V A F A I Y Y I P L 5 6140 191 A I Y Y I P L I S L 5 6141 195 I P L I S L S I V H 5 6142 208 K Q A P A Y V H T M 5 6143 228 L M N P V I Y S V K 5 6144 16 S I S T L V T M L S 4 6145 34 I S F N A C L S H M 4 6146 38 A C L S H M F F I K 4 6147 40 L S H M F F I K F F 4 6148 43 M F F I K F F T V M 4 6149 53 E S S V L L A M A F 4 6150 59 A M A F D R F V A V 4 6151 79 T D S R I A Q I G V 4 6152 90 S V I R G L L M L T 4 6153 99 T P M V A L L I R L 4 6154 112 H S Q V L H H S Y C 4 6155 140 V G L T A M F S T V 4 6156 158 L S Y V L I I R T V 4 6157 159 S Y V L I I R T V L 4 6158 160 Y V L I I R T V L S 4 6159 168 L S V A S P E E R K 4 6160 169 S V A S P E E R K E 4 6161 198 I S L S I V H R F G 4 6162 199 S L S I V H R F G K 4 6163 211 P A Y V H T M I A N 4 6164 218 I A N T Y L L I S P 4 6165 7 M L S A T D L G L S 3 6166 12 D L G L S I S T L V 3 6167 25 S I F W F N V R E I 3 6168 30 N V R E I S F N A C 3 6169 35 S F N A C L S H M F 3 6170 42 H M F F I K F F T V 3 6171 64 R F V A V S N P L R 3 6172 81 S R I A Q I G V A S 3 6173 87 G V A S V I R G L L 3 6174 96 L M L T P M V A L L 3 6175 119 S Y C Y H P D V M K 3 6176 134 T R I N S A V G L T 3 6177 138 S A V G L T A M F S 3 6178 145 M F S T V G V D L L 3 6179 154 L L I L L S Y V L I 3 6180 174 E E R K E T F S T C 3 6181 189 A F A I Y Y I P L I 3 6182 201 S I V H R F G K Q A 3 6183 222 Y L L I S P L M N P 3 6184 244 A V I K I L H S K E 3 6185 2 Y Y F L S M L S A T 2 6186 3 Y F L S M L S A T D 2 6187 4 F L S M L S A T D L 2 6188 9 S A T D L G L S I S 2 6189 32 R E I S F N A C L S 2 6190 41 S H M F F I K E F T 2 6191 44 F F I K F F T V M E 2 6192 45 F I K F F T V M E S 2 6193 48 F F T V M E S S V L 2 6194 55 S V L L A M A F D R 2 6195 58 L A M A F D R F V A 2 6196 60 M A F D R F V A V S 2 6197 62 F D R F V A V S N P 2 6198 67 A V S N P L R Y A M 2 6199 69 S N P L R Y A M I L 2 6200 74 Y A M I L T D S R I 2 6201 84 A Q I G V A S V I R 2 6202 94 G L L M L T P M V A 2 6203 100 P M V A L L I R L S 2 6204 103 A L L I R L S Y C H 2 6205 107 R L S Y C H S Q V L 2 6206 110 Y C H S Q V L H H S 2 6207 115 V L H H S Y C Y H P 2 6208 130 S C T D T R I N S A 2 6209 141 G L T A M F S T V G 2 6210 143 T A M F S T V G V D 2 6211 144 A M F S T V G V D L 2 6212 153 L L L I L L S Y V L 2 6213 167 V L S V A S P E E R 2 6214 192 I Y Y I P L I S L S 2 6215 194 Y I P L I S L S I V 2 6216 206 F G K Q A P A Y V H 2 6217 214 V H T M I A N T Y L 2 6218 229 M N P V I Y S V K T 2 6219 231 P V I Y S V K T K Q 2 6220 235 S V K T K Q I R R A 2 6221 236 V K T K Q I R R A V 2 6222 11 T D L G L S I S T L 1 6223 14 G L S I S T L V T M 1 6224 26 I F W F N V R E I S 1 6225 29 F N V R E I S F N A 1 6226 36 F N A C L S H M F F 1 6227 52 M E S S V L L A M A 1 6228 57 L L A M A F D R F V 1 6229 66 V A V S N P L R Y A 1 6230 75 A M I L T D S R I A 1 6231 77 I L T D S R I A Q I 1 6232 82 R I A Q I G V A S V 1 6233 83 I A Q I G V A S V I 1 6234 93 R G L L M L T P M V 1 6235 95 L L M L T P M V A L 1 6236 102 V A L L I R L S Y C 1 6237 104 L L I R L S Y C H S 1 6238 126 V M K L S C T D T R 1 6239 128 K L S C T D T R I N 1 6240 132 T D T R I N S A V G 1 6241 139 A V G L T A M F S T 1 6242 152 D L L L I L L S Y V 1 6243 162 L I I R T V L S V A 1 6244 164 I R T V L S V A S P 1 6245 182 T C V S H I V A F A 1 6246 196 P L I S L S I V H R 1 6247 197 L I S L S I V H R F 1 6248 203 V H R F G K Q A P A 1 6249 209 Q A P A Y V H T M I 1 6250 210 A P A Y V H T M I A 1 6251 212 A Y V H T M I A N T 1 6252 219 A N T Y L L I S P L 1 6253 223 L L I S P L M N P V 1 6254 227 P L M N P V I Y S V 1 6255 230 N P V I Y S V K T K 1 6256 232 V I Y S V K T K Q I 1 6257 245 V I K I L H S K E T 1 6258 HLA-A26 10-mers v.1: 238P1B2 133 D T R I N S A V G L 26 6259 181 S T C V S H I V A F 26 6260 18 S T L V T M L S I F 25 6261 178 E T F S T C V S H I 25 6262 197 L I S L S I V H R F 25 6263 20 L V T M L S I F W F 24 6264 39 C L S H M F F I K F 23 6265 56 V L L A M A F D R F 23 6266 191 A I Y Y I P L I S L 23 6267 65 F V A V S N P L R Y 22 6268 95 L L M L T P M V A L 22 6269 101 M V A L L I R L S Y 22 6270 148 T V G V D L L L I L 22 6271 240 Q I R R A V I K I L 22 6272 14 G L S I S T L V T M 21 6273 43 M F F I K F F T V M 20 6274 87 G V A S V I R G L L 20 6275 213 Y V H T M I A N T Y 20 6276 49 F T V M E S S V L L 19 6277 67 A V S N P L R Y A M 19 6278 152 D L L L I L L S Y V 19 6279 220 N T Y L L I S P L M 19 6280 53 E S S V L L A M A F 18 6281 82 R I A Q I G V A S V 18 6282 145 M F S T V G V D L L 18 6283 215 H T M I A N T Y L L 18 6284 25 S I F W F N V R E I 17 6285 30 N V R E I S F N A C 17 6286 33 E I S F N A C L S H 17 6287 35 S F N A C L S H M F 17 6288 45 F I K F F T V M E S 17 6289 90 S V I R G L L M L T 17 6290 107 R L S Y C H S Q V L 17 6291 125 D V M K L S C T D T 17 6292 162 L I I R T V L S V A 17 6293 196 P L I S L S I V H R 17 6294 4 F L S M L S A T D L 16 6295 15 L S I S T L V T M L 16 6296 34 I S F N A C L S H M 16 6297 50 T V M E S S V L L A 16 6298 63 D R F V A V S N P L 16 6299 91 V I R G L L M L T P 16 6300 147 S T V G V D L L L I 16 6301 153 L L L I L L S Y V L 16 6302 194 Y I P L I S L S I V 16 6303 204 H R F G K Q A P A Y 16 6304 235 S V K T K Q I R R A 16 6305 11 T D L G L S I S T L 15 6306 48 F F T V M E S S V L 15 6307 77 I L T D S R I A Q I 15 6308 85 Q I G V A S V I R G 15 6309 98 L T P M V A L L I R 15 6310 104 L L I R L S Y C H S 15 6311 139 A V G L T A M F S T 15 6312 150 G V D L L L I L L S 15 6313 151 V D L L L I L L S Y 15 6314 154 L L I L L S Y V L I 15 6315 161 V L I I R T V L S V 15 6316 184 V S H I V A F A I Y 15 6317 187 I V A F A I Y Y I P 15 6318 208 K Q A P A Y V H T M 15 6319 217 M I A N T Y L L I S 15 6320 223 L L I S P L M N P V 15 6321 244 A V I K I L H S K E 15 6322 12 D L G L S I S T L V 14 6323 21 V T M L S I F W F N 14 6324 40 L S H M F F I K F F 14 6325 51 V M E S S V L L A M 14 6326 114 Q V L H H S Y C Y H 14 6327 120 Y C Y H P D V M K L 14 6328 155 L I L L S Y V L I I 14 6329 186 H I V A F A I Y Y I 14 6330 188 V A F A I Y Y I P L 14 6331 222 Y L L I S P L M N P 14 6332 27 F W F N V R E I S F 13 6333 78 L T D S R I A Q I G 13 6334 86 I G V A S V I R G L 13 6335 89 A S V I R G L L M L 13 6336 96 L M L T P M V A L L 13 6337 99 T P M V A L L I R L 13 6338 131 C T D T R I N S A V 13 6339 136 I N S A V G L T A M 13 6340 137 N S A V G L T A M F 13 6341 142 L T A M F S T V G V 13 6342 149 V G V D L L L I L L 13 6343 156 I L L S Y V L I I R 13 6344 157 L L S Y V L I I R T 13 6345 166 T V L S V A S P E E 13 6346 169 S V A S P E E R K E 13 6347 171 A S P E E R K E T F 13 6348 219 A N T Y L L I S P L 13 6349 224 L I S P L M N P V I 13 6350 227 P L M N P V I Y S V 13 6351 231 P V I Y S V K T K Q 13 6352 237 K T K Q I R R A V I 13 6353 7 M L S A T D L G L S 12 6354 10 A T D L G L S I S T 12 6355 23 M L S I F W F N V R 12 6356 36 F N A C L S H M F F 12 6357 71 P L R Y A M I L T D 12 6358 92 I R G L L M L T P M 12 6359 111 C H S Q V L H H S Y 12 6360 144 A M F S T V G V D L 12 6361 163 I I R T V L S V A S 12 6362 174 E E R K E T F S T C 12 6363 185 S H I V A F A I Y Y 12 6364 189 A F A I Y Y I P L I 12 6365 202 I V H R F G K Q A P 12 6366 225 I S P L M N P V I Y 12 6367 232 V I Y S V K T K Q I 12 6368 245 V I K I L H S K E T 12 6369 16 S I S T L V T M L S 11 6370 28 W F N V R E I S F N 11 6371 44 F F I K F F T V M E 11 6372 76 M I L T D S R I A Q 11 6373 105 L I R L S Y C H S Q 11 6374 135 R I N S A V G L T A 11 6375 160 Y V L I I R T V L S 11 6376 165 R T V L S V A S P E 11 6377 183 C V S H I V A F A I 11 6378 201 S I V H R F G K Q A 11 6379 6 S M L S A T D L G L 10 6380 47 K F F T V M E S S V 10 6381 55 S V L L A M A F D R 10 6382 69 S N P L R Y A M I L 10 6383 80 D S R I A Q I G V A 10 6384 97 M L T P M V A L L I 10 6385 113 S Q V L H H S Y C Y 10 6386 31 V R E I S F N A C L 9 6387 57 L L A M A F D R F V 9 6388 61 A F D R F V A V S N 9 6389 94 G L L M L T P M V A 9 6390 115 V L H H S Y C Y H P 9 6391 167 V L S V A S P E E R 9 6392 175 E R K E T F S T C V 9 6393 199 S L S I V H R F G K 9 6394 2 Y Y F L S M L S A T 8 6395 3 Y F L S M L S A T D 8 6396 19 T L V T M L S I F W 8 6397 60 M A F D R F V A V S 8 6398 68 V S N P L R Y A M I 8 6399 88 V A S V I R G L L M 8 6400 103 A L L I R L S Y C H 8 6401 118 H S Y C Y H P D V M 8 6402 128 K L S C T D T R I N 8 6403 141 G L T A M F S T V G 8 6404 146 F S T V G V D L L L 8 6405 159 S Y V L I I R T V L 8 6406 205 R F G K Q A P A Y V 8 6407 214 V H T M I A N T Y L 8 6408 46 I K F F T V M E S S 7 6409 52 M E S S V L L A M A 7 6410 59 A M A F D R F V A V 7 6411 64 R F V A V S N P L R 7 6412 200 L S I V H R F G K Q 7 6413 228 L M N P V I Y S V K 7 6414 239 K Q I R R A V I K I 7 6415 9 S A T D L G L S I S 6 6416 26 I F W F N V R E I S 6 6417 42 H M F F I K F F T V 6 6418 62 F D R F V A V S N P 6 6419 110 Y C H S Q V L H H S 6 6420 121 C Y H P D V M K L S 6 6421 123 H P D V M K L S C T 6 6422 130 S C T D T R I N S A 6 6423 134 T R I N S A V G L T 6 6424 164 I R T V L S V A S P 6 6425 179 T F S T C V S H I V 6 6426 182 T C V S H I V A F A 6 6427 207 G K Q A P A Y V H T 6 6428 212 A Y V H T M I A N T 6 6429 216 T M I A N T Y L L I 6 6430 242 R R A V I K I L H S 6 6431 1 M Y Y F L S M L S A 5 6432 17 I S T L V T M L S I 5 6433 38 A C L S H M F F I K 5 6434 54 S S V L L A M A F D 5 6435 66 V A V S N P L R Y A 5 6436 84 A Q I G V A S V I R 5 6437 102 V A L L I R L S Y C 5 6438 109 S Y C H S Q V L H H 5 6439 116 L H H S Y C Y H P D 5 6440 140 V G L T A M F S T V 5 6441 192 I Y Y T P L I S L S 5 6442 193 Y Y I P L I S L S I 5 6443 211 P A Y V H T M I A N 5 6444 218 I A N T Y L L I S P 5 6445 243 R A V I K I L H S K 5 6446 24 L S I F W P N V R E 4 6447 37 N A C L S H M F F I 4 6448 70 N P L R Y A M I L T 4 6449 72 L R Y A M I L T D S 4 6450 81 S R I A Q I G V A S 4 6451 100 P M V A L L I R L S 4 6452 143 T A M F S T V G V D 4 6453 158 L S Y V L I I R T V 4 6454 226 S P L M N P V I Y S 4 6455 230 N P V I Y S V K T K 4 6456 32 R E I S F N A C L S 3 6457 106 I R L S Y C H S Q V 3 6458 122 Y H P D V M K L S C 3 6459 138 S A V G L T A M F S 3 6460 170 V A S P E E R K E T 3 6461 229 M N P V I Y S V K T 3 6462 8 L S A T D L G L S I 2 6463 22 T M L S I F W F N V 2 6464 73 R Y A M I L T D S R 2 6465 74 Y A M I L T D S R I 2 6466 75 A M I L T D S R I A 2 6467 93 R G L L M L T P M V 2 6468 119 S Y C Y H P D V M K 2 6469 127 M K L S C T D T R I 2 6470 172 S P E E R K E T F S 2 6471 177 K E T F S T C V S H 2 6472 190 F A I Y Y I P L I S 2 6473 206 F G K Q A P A Y V H 2 6474 209 Q A P A Y V H T M I 2 6475 210 A P A Y V H T M I A 2 6476 234 Y S V K T K Q I R R 2 6477 236 V K T K Q I R R A V 2 6478 13 L G L S I S T L V T 1 6479 29 F N V R E I S F N A 1 6480 79 T D S R I A Q I G V 1 6481 83 I A Q I G V A S V I 1 6482 108 L S Y C H S Q V L H 1 6483 112 H S Q V L H H S Y C 1 6484 117 H H S Y C Y H P D V 1 6485 126 V M K L S C T D T R 1 6486 129 L S C T D T R I N S 1 6487 132 T D T R I N S A V G 1 6488 168 L S V A S P E E R K 1 6489 173 P E E R K E T F S T 1 6490 198 I S L S I V H R F G 1 6491 203 V H R F G K Q A P A 1 6492 221 T Y L L I S P L M N 1 6493 233 I Y S V K T K Q I R 1 6494 238 T K Q I R R A V I K 1 6495 HLA-A3 10-mers v.1: 238P1B2 101 M V A L L I R L S Y 24 6496 199 S L S I V H R F G K 23 6497 213 Y V H T M I A N T Y 23 6498 135 R I N S A V G L T A 22 6499 55 S V L L A M A F D R 21 6500 65 F V A V S N P L R Y 21 6501 162 L I I R T V L S V A 21 6502 196 P L I S L S I V H R 21 6503 71 P L R Y A M I L T D 20 6504 91 V I R G L L M L T P 20 6505 103 A L L I R L S Y C H 20 6506 228 L M N P V I Y S V K 20 6507 14 G L S I S T L V T M 19 6508 23 M L S I F W F N V R 19 6509 77 I L T D S R I A Q I 19 6510 97 M L T P M V A L L I 19 6511 161 V L I I R T V L S V 19 6512 244 A V I K I L H S K E 19 6513 56 V L L A M A F D R F 18 6514 82 R I A Q I G V A S V 18 6515 90 S V I R G L L M L T 18 6516 94 G L L M L T P M V A 18 6517 107 R L S Y C H S Q V L 18 6518 114 Q V L H H S Y C Y H 18 6519 154 L L I L L S Y V L I 18 6520 156 I L L S Y V L I I R 18 6521 163 I I R T V L S V A S 18 6522 238 T K Q I R R A V I K 18 6523 33 E I S F N A C L S H 17 6524 141 G L T A M F S T V G 17 6525 153 L L L I L L S Y V L 17 6526 160 Y V L I I R T V L S 17 6527 191 A I Y Y I P L I S L 17 6528 243 R A V I K I L H S K 17 6529 67 A V S N P L R Y A M 16 6530 84 A Q I G V A S V I R 16 6531 95 L L M L T P M V A L 16 6532 104 L L I R L S Y C H S 16 6533 119 S Y C Y H P D V M K 16 6534 139 A V G L T A M F S T 16 6535 152 D L L L I L L S Y V 16 6536 202 I V H R F C K Q A P 16 6537 240 Q I R R A V I K I L 16 6538 20 L V T M L S I F W F 15 6539 30 N V R E I S F N A C 15 6540 38 A C L S H M F F I K 15 6541 61 A F D R F V A V S N 15 6542 167 V L S V A S P E E R 15 6543 201 S I V H R F G K Q A 15 6544 224 L I S P L M N P V I 15 6545 230 N P V I Y S V K T K 15 6546 232 V I Y S V K T K Q I 15 6547 4 F L S M L S A T D L 14 6548 39 C L S H M F F I K F 14 6549 50 T V M E S S V L L A 14 6550 81 S R I A Q I G V A S 14 6551 105 L I R L S Y C H S Q 14 6552 150 G V D L L L I L L S 14 6553 151 V D L L L I L L S Y 14 6554 155 L I L L S Y V L I I 14 6555 183 C V S H I V A F A I 14 6556 222 Y L L I S P L M N P 14 6557 225 I S P L M N P V I Y 14 6558 231 P V I Y S V K T K Q 14 6559 7 M L S A T D L G L 5 13 6560 83 I A Q I G V A S V I 13 6561 87 G V A S V I R G L L 13 6562 128 K L S C T D T R I N 13 6563 148 T V G V D L L L I L 13 6564 177 K E T F S T C V S H 13 6565 185 S H I V A F A I Y Y 13 6566 187 I V A F A I Y Y I P 13 6567 195 I P L I S L S I V H 13 6568 197 L I S L S I V H R F 13 6569 217 M I A N T Y L L I S 13 6570 223 L L I S P L M N P V 13 6571 227 P L M N P V I Y S V 13 6572 12 D L G L S I S T L V 12 6573 7 M I L T D S R I A Q 12 6574 108 L S Y C H S Q V L H 12 6575 166 T V L S V A S P E E 12 6576 168 L S V A S P E E R K 12 6577 169 S V A S P E E R K E 12 6578 171 A S P E E R K E T F 12 6579 194 Y I P L I S L S I V 12 6580 204 H R F G K Q A P A Y 12 6581 206 F G K Q A P A Y V H 12 6582 235 S V K T K Q I R R A 12 6583 237 K T K Q I R R A V I 12 6584 11 T D L G L S I S T L 11 6585 57 L L A M A F D R F V 11 6586 73 R Y A M I L T D S R 11 6587 125 D V M K L S C T D T 11 6588 133 D T R I N S A V G L 11 6589 165 R T V L S V A S P E 11 6590 193 Y Y I P L I S L S I 11 6591 208 K Q A P A Y V H T M 11 6592 241 I R R A V I K I L H 11 6593 16 S I S T L V T M L S 10 6594 18 S T L V T M L S I F 10 6595 19 T L V T M L S I F W 10 6596 32 R E I S F N A C L S 10 6597 45 F I K F F T V M E S 10 6598 64 R F V A V S N P L R 10 6599 85 Q I G V A S V I R G 10 6600 106 I R L S Y C H S Q V 10 6601 115 V L H H S Y C Y H P 10 6602 126 V M K L S C T D T R 10 6603 132 T D T R I N S A V G 10 6604 144 A M F S T V G V D L 10 6605 157 L L S Y V L I I R T 10 6606 174 E E R K E T F S T C 10 6607 181 S T C V S H I V A F 10 6608 184 V S H I V A F A I Y 10 6609 186 H I V A F A I Y Y I 10 6610 192 I Y Y I P L I S L S 10 6611 245 V I K I L H S K E T 10 6612 1 M Y Y F L S M L S A 9 6613 3 Y F L S M L S A T D 9 6614 8 L S A T D L G L S I 9 6615 13 L G L S I S T L V T 9 6616 25 S I F W F N V R E I 9 6617 53 E S S V L L A M A F 9 6618 59 A M A F D R F V A V 9 6619 80 D S R I A Q I G V A 9 6620 118 H S Y C Y H P D V M 9 6621 137 N S A V G L T A M F 9 6622 140 V G L T A M F S T V 9 6623 239 K Q I R R A V I K I 9 6624 17 I S T L V T M L S I 8 6625 35 S F N A C L S H M F 8 6626 44 F F I K F F T V M E 8 6627 47 K F F T V M E S S V 8 6628 60 M A F D R F V A V S 8 6629 89 A S V I R G L L M L 8 6630 93 R G L L M L T P M V 8 6631 109 S Y C H S Q V L H H 8 6632 111 C H S Q V L H H S Y 8 6633 113 S Q V L H H S Y C Y 8 6634 158 L S Y V L I I R T V 8 6635 159 S Y V L I I R T V L 8 6636 176 R K E T F S T C V S 8 6637 205 R F G K Q A P A Y V 8 6638 216 T M I A N T Y L L I 8 6639 221 T Y L L I S P L M N 8 6640 242 R R A V I K I L H S 8 6641 24 L S I F W F N V R E 7 6642 27 F W F N V R E I S F 7 6643 48 F F T V M E S S V L 7 6644 58 L A M A F D R F V A 7 6645 68 V S N P L R Y A M I 7 6646 72 L R Y A M I L T D S 7 6647 75 A M I L T D S R I A 7 6648 98 L T P M V A L L I R 7 6649 138 S A V G L T A M F S 7 6650 164 I R T V L S V A S P 7 6651 190 F A I Y Y I P L I S 7 6652 6 S M L S A T D L G L 6 6653 10 A T D L G L S I S T 6 6654 34 I S F N A C L S H M 6 6655 36 F N A C L S H M F F 6 6656 43 M F F I K F F T V M 6 6657 62 F D R F V A V S N P 6 6658 69 S N P L R Y A M I L 6 6659 88 V A S V I R G L L M 6 6660 102 V A L L I R L S Y C 6 6661 122 Y H P D V M K L S C 6 6662 136 I N S A V G L T A M 6 6663 147 S T V G V D L L L I 6 6664 203 V H R F G K Q A P A 6 6665 207 G K Q A P A Y V H T 6 6666 209 Q A P A Y V H T M I 6 6667 212 A Y V H T M I A N T 6 6668 219 A N T Y L L I S P L 6 6669 233 I Y S V K T K Q I R 6 6670 2 Y Y F L S M L S A T 5 6671 9 S A T D L G L S I S 5 6672 15 L S I S T L V T M L 5 6673 54 S S V L L A M A F D 5 6674 70 N P L R Y A M I L T 5 6675 96 L M L T P M V A L L 5 6676 120 Y C Y H P D V M K L 5 6677 134 T R I N S A V G L T 5 6678 142 L T A M F S T V G V 5 6679 143 T A M F S T V G V D 5 6680 173 P E E R K E T F S T 5 6681 189 A F A I Y Y I P L I 5 6682 210 A P A Y V H T M I A 5 6683 211 P A Y V H T M I A N 5 6684 220 N T Y L L I S P L M 5 6685 31 V R E I S F N A C L 4 6686 40 L S H M F F I K F F 4 6687 42 H M F F I K F F T V 4 6688 46 I K F F T V M E S S 4 6689 49 F T V M E S S V L L 4 6690 51 V M E S S V L L A M 4 6691 52 M E S S V L L A M A 4 6692 74 Y A M I L T D S R I 4 6693 86 I G V A S V I R G L 4 6694 92 I R G L L M L T P M 4 6695 124 P D V M K L S C T D 4 6696 130 S C T D T R I N S A 4 6697 131 C T D T R I N S A V 4 6698 170 V A S P E E R K E T 4 6699 180 F S T C V S H I V A 4 6700 198 I S L S I V H R F G 4 6701 200 L S I V H R F G K Q 4 6702 218 I A N T Y L L I S P 4 6703 229 M N P V I Y S V K T 4 6704 234 Y S V K T K Q I R R 4 6705 26 I F W F N V R E I S 3 6706 28 W F N V R E I S F N 3 6707 29 F N V R E I S F N A 3 6708 78 L T D S R I A Q I G 3 6709 79 T D S R I A Q I G V 3 6710 112 H S Q V L H H S Y C 3 6711 121 C Y H P D V M K L S 3 6712 123 H P D V M K L S C T 3 6713 127 M K L S C T D T R I 3 6714 146 F S T V G V D L L L 3 6715 172 S P E E R K E T F S 3 6716 175 E R K E T F S T C V 3 6717 178 E T F S T C V S H I 3 6718 182 T C V S H I V A F A 3 6719 226 S P L M N P V I Y S 3 6720 22 T M L S I F W F N V 2 6721 100 P M V A L L I R L S 2 6722 188 V A F A I Y Y I P L 2 6723 236 V K T K Q I R R A V 2 6724 5 L S M L S A T D L G 1 6725 21 V T M L S I F W F N 1 6726 37 N A C L S H M F F I 1 6727 41 S H M F F I K F F T 1 6728 129 L S C T D T R I N S 1 6729 149 V G V D L L L I L L 1 6730 214 V H T M I A N T Y L 1 6731 215 H T M I A N T Y L L 1 6732 HLA-B*0702 10-mers v.1: 238P1B2 99 T P M V A L L I R L 21 6733 210 A P A Y V H T M I A 20 6734 123 H P D V M K L S C T 17 6735 70 N P L R Y A M I L T 16 6736 89 A S V I R G L L M L 14 6737 95 L L M L T P M V A L 14 6738 107 R L S Y C H S Q V L 14 6739 133 D T R I N S A V G L 14 6740 144 A M F S T V G V D L 14 6741 4 F L S M L S A T D L 13 6742 59 A M A F D R F V A V 13 6743 145 M F S T V G V D L L 13 6744 148 T V G V D L L L I L 13 6745 159 S Y V L I I R T V L 13 6746 219 A N T Y L L I S P L 13 6747 6 S M L S A T D L G L 12 6748 11 T D L G L S I S T L 12 6749 15 L S I S T L V T M L 12 6750 49 F T V M E S S V L L 12 6751 67 A V S N P L R Y A M 12 6752 86 I G V A S V I R G L 12 6753 87 G V A S V I R G L L 12 6754 96 L M L T P M V A L L 12 6755 136 I N S A V G L T A M 12 6756 146 F S T V G V D L L L 12 6757 172 S P E E R K E T F S 12 6758 188 V A F A I Y Y I P L 12 6759 191 A I Y Y I P L I S L 12 6760 195 I P L I S L S I V H 12 6761 215 H T M I A N T Y L L 12 6762 240 Q I R R A V I K I L 12 6763 31 V R E I S F N A C L 11 6764 48 F F T V M E S S V L 11 6765 63 D R F V A V S N P L 11 6766 120 Y C Y H P D V M K L 11 6767 153 L L L I L L S Y V L 11 6768 214 V H T M I A N T Y L 11 6769 224 L I S P L M N P V I 11 6770 226 S P L M N P V I Y S 11 6771 230 N P V I Y S V K T K 11 6772 13 L G L S I S T L V T 10 6773 14 G L S I S T L V T M 10 6774 50 T V M E S S V L L A 10 6775 53 E S S V L L A M A F 10 6776 58 L A M A F D R F V A 10 6777 69 S N P L R Y A M I L 10 6778 82 R I A Q I G V A S V 10 6779 88 V A S V I R G L L M 10 6780 92 I R G L L M L T P M 10 6781 142 L T A M F S T V G V 10 6782 147 S T V G V D L L L I 10 6783 149 V G V D L L L I L L 10 6784 170 V A S P E E R K E T 10 6785 189 A F A I Y Y I P L I 10 6786 203 V H R F G K Q A P A 10 6787 205 R F G K Q A P A Y V 10 6788 208 K Q A P A Y V H T M 10 6789 237 K T K Q I R R A V I 10 6790 8 L S A T D L G L S I 9 6791 10 A T D L G L S I S T 9 6792 17 I S T L V T M L S I 9 6793 39 C L S H M F F I K F 9 6794 43 M F F I K F F T V M 9 6795 51 V M E S S V L L A M 9 6796 52 M E S S V L L A M A 9 6797 80 D S R I A Q I G V A 9 6798 93 R G L L M L T P M V 9 6799 97 M L T P M V A L L I 9 6800 117 H H S Y C Y H P D V 9 6801 125 D V M K L S C T D T 9 6802 135 R I N S A V G L T A 9 6803 137 N S A V G L T A M F 9 6804 139 A V G L T A M F S T 9 6805 155 L I L L S Y V L I I 9 6806 161 V L I I R T V L S V 9 6807 181 S T C V S H I V A F 9 6808 182 T C V S H I V A F A 9 6809 183 C V S H I V A F A I 9 6810 193 Y Y I P L I S L S I 9 6811 197 L I S L S I V H R F 9 6812 207 G K Q A P A Y V H T 9 6813 212 A Y V H T M I A N T 9 6814 216 T M I A N T Y L L I 9 6815 236 V K T K Q I R R A V 9 6816 1 M Y Y F L S M L S A 8 6817 12 D L G L S I S T L V 8 6818 36 F N A C L S H M F F 8 6819 41 S H M F F I K F F T 8 6820 56 V L L A M A F D R F 8 6821 57 L L A M A F D R F V 8 6822 68 V S N P L R Y A M I 8 6823 75 A M I L T D S R I A 8 6824 77 I L T D S R I A Q I 8 6825 79 T D S R I A Q I G V 8 6826 83 I A Q I G V A S V I 8 6827 131 C T D T R I N S A V 8 6828 154 L L I L L S Y V L I 8 6829 157 L L S Y V L I I R T 8 6830 171 A S P E E R K E T F 8 6831 175 E R K E T F S T C V 8 6832 178 E T F S T C V S H I 8 6833 179 T F S T C V S H I V 8 6834 209 Q A P A Y V H T M I 8 6835 229 M N P V I Y S V K T 8 6836 239 K Q I R R A V I K I 8 6837 2 Y Y F L S M L S A T 7 6838 25 S I F W F N V R E I 7 6839 34 I S F N A C L S H M 7 6840 35 S F N A C L S H M F 7 6841 37 N A C L S H M F F I 7 6842 40 L S H M F F I K F F 7 6843 42 H M F F I K F F T V 7 6844 47 K F F T V M E S S V 7 6845 74 Y A M I L T D S R I 7 6846 94 G L L M L T P M V A 7 6847 106 I R L S Y C H S Q V 7 6848 118 H S Y C Y H P D V M 7 6849 127 M K L S C T D T R I 7 6850 134 T R I N S A V G L T 7 6851 140 V G L T A M F S T V 7 6852 162 L I I R T V L S V A 7 6853 173 P E E R K E T F S T 7 6854 180 F S T C V S H I V A 7 6855 186 H I V A F A I Y Y I 7 6856 220 N T Y L L I S P L M 7 6857 223 L L I S P L M N P V 7 6858 227 P L M N P V I Y S V 7 6859 232 V I Y S V K T K Q I 7 6860 18 S T L V T M L S I F 6 6861 20 L V T M L S I F W F 6 6862 22 T M L S I F W F N V 6 6863 27 F W F N V R E I S F 6 6864 29 F N V R E I S F N A 6 6865 66 V A V S N P L R Y A 6 6866 84 A Q I G V A S V I R 6 6867 90 S V I R G L L M L T 6 6868 91 V I R G L L M L T P 6 6869 130 S C T D T R I N S A 6 6870 152 D L L L I L L S Y V 6 6871 158 L S Y V L I I R T V 6 6872 163 I I R T V L S V A S 6 6873 194 Y I P L I S L S I V 6 6874 201 S I V H R F G K Q A 6 6875 235 S V K T K Q I R R A 6 6876 245 V I K I L H S K E T 6 6877 33 E I S F N A C L S H 5 6878 61 A F D R F V A V S N 5 6879 174 E E R K E T F S T C 5 6880 23 M L S I F W F N V R 4 6881 30 N V R E I S F N A C 4 6882 44 F F I K F F T V M E 4 6883 71 P L R Y A M I L T D 4 6884 81 S R I A Q I G V A S 4 6885 101 M V A L L I R L S Y 4 6886 128 K L S C T D T R I N 4 6887 204 H R F G K Q A P A Y 4 6888 241 I R R A V I K I L H 4 6889 242 R R A V I K I L H S 4 6890 7 M L S A T D L G L S 3 6891 16 S I S T L V T M L S 3 6892 21 V T M L S I F W F N 3 6893 38 A C L S H M F F I K 3 6894 60 M A F D R F V A V S 3 6895 62 F D R F V A V S N P 3 6896 64 R F V A V S N P L R 3 6897 65 F V A V S N P L R Y 3 6898 73 R Y A M I L T D S R 3 6899 109 S Y C H S Q V L H H 3 6900 138 S A V G L T A M F S 3 6901 141 G L T A M F S T V G 3 6902 143 T A M F S T V G V D 3 6903 150 G V D L L L I L L S 3 6904 160 Y V L I I R T V L S 3 6905 164 I R T V L S V A S P 3 6906 176 R K E T F S T C V S 3 6907 177 K E T F S T C V S H 3 6908 199 S L S I V H R F G K 3 6909 217 M I A N T Y L L I S 3 6910 225 I S P L M N P V I Y 3 6911 233 I Y S V K T K Q I R 3 6912 244 A V I K I L H S K E 3 6913 5 L S M L S A T D L G 2 6914 24 L S I F W F N V R E 2 6915 26 I F W F N V R E I S 2 6916 54 S S V L L A M A F D 2 6917 72 L R Y A M I L T D S 2 6918 76 M I L T D S R I A Q 2 6919 85 Q I G V A S V I R G 2 6920 98 L T P M V A L L I R 2 6921 103 A L L I R L S Y C H 2 6922 105 L I R L S Y C H S Q 2 6923 108 L S Y C H S Q V L H 2 6924 111 C H S Q V L H H S Y 2 6925 119 S Y C Y H P D V M K 2 6926 122 Y H P D V M K L S C 2 6927 132 T D T R I N S A V G 2 6928 151 V D L L L I L L S Y 2 6929 165 R T V L S V A S P E 2 6930 167 V L S V A S P E E R 2 6931 184 V S H I V A F A I Y 2 6932 187 I V A F A I Y Y I P 2 6933 190 F A I Y Y I P L I S 2 6934 196 P L I S L S I V H R 2 6935 198 I S L S I V H R F G 2 6936 202 I V H R F G K Q A P 2 6937 221 T Y L L I S P L M N 2 6938 228 L M N P V I Y S V K 2 6939 238 T K Q I R R A V I K 2 6940 3 Y F L S M L S A T D 1 6941 19 T L V T M L S I F W 1 6942 28 W F N V R E I S F N 1 6943 32 R E I S F N A C L S 1 6944 45 F I K F F T V M E S 1 6945 46 I K F F T V M E S S 1 6946 78 L T D S R I A Q I G 1 6947 100 P M V A L L I R L S 1 6948 116 L H H S Y C Y H P D 1 6949 121 C Y H P D V M K L S 1 6950 126 V M K L S C T D T R 1 6951 129 L S C T D T R I N S 1 6952 156 I L L S Y V L I I R 1 6953 166 T V L S V A S P E E 1 6954 168 L S V A S P E E R K 1 6955 169 S V A S P E E R K E 1 6956 192 I Y Y I P L I S L S 1 6957 200 L S I V H R F G K Q 1 6958 206 F G K Q A P A Y V H 1 6959 211 P A Y V H T M I A N 1 6960 218 I A N T Y L L I S P 1 6961 231 P V I Y S V K T K Q 1 6962 234 Y S V K T K Q I R R 1 6963 243 R A V I K I L H S K 1 6964 HLA-B*4402 10-mers v.1: 238P1B2 181 S T C V S H I V A F 17 6965 11 T D L G L S I S T L 16 6966 53 E S S V L L A M A F 16 6967 144 A M F S T V G V D L 16 6968 185 S H I V A F A I Y Y 16 6969 204 H R F G K Q A P A Y 16 6970 219 A N T Y L L I S P L 16 6971 239 K Q I R R A V I K I 16 6972 240 Q I R R A V I K I L 16 6973 32 R E I S F N A C L S 15 6974 40 L S H M F F I K F F 15 6975 89 A S V I R G L L M L 15 6976 95 L L M L T P M V A L 15 6977 171 A S P E E R K E T F 15 6978 189 A F A I Y Y I P L I 15 6979 191 A I Y Y I P L I S L 15 6980 193 Y Y I P L I S L S I 15 6981 15 L S I S T L V T M L 14 6982 27 F W F N V R E I S F 14 6983 39 C L S H M F F I K F 14 6984 69 S N P L R Y A M I L 14 6985 86 I G V A S V I R G L 14 6986 96 L M L T P M V A L L 14 6987 97 M L T P M V A L L I 14 6988 145 M F S T V G V D L L 14 6989 149 V G V D L L L I L L 14 6990 151 V D L L L I L L S Y 14 6991 154 L L I L L S Y V L I 14 6992 159 S Y V L I I R T V L 14 6993 174 E E R K E T F S T C 14 6994 197 L I S L S I V H R F 14 6995 213 Y V H T M I A N T Y 14 6996 225 I S P L M N P V I Y 14 6997 6 S M L S A T D L C L 13 6998 18 S T L V T M L S I F 13 6999 20 L V T M L S I F W F 13 7000 25 S I F W F N V R E I 13 7001 63 D R F V A V S N P L 13 7002 77 I L T D S R I A Q I 13 7003 99 T P M V A L L I R L 13 7004 101 M V A L L I R L S Y 13 7005 147 S T V G V D L L L I 13 7006 178 E T F S T C V S H I 13 7007 188 V A F A I Y Y I P L 13 7008 215 H T M I A N T Y L L 13 7009 216 T M I A N T Y L L I 13 7010 224 L I S P L M N P V I 13 7011 4 F L S M L S A T D L 12 7012 19 T L V T M L S I F W 12 7013 35 S F N A C L S H M F 12 7014 49 F T V M E S S V L L 12 7015 52 M E S S V L L A M A 12 7016 56 V L L A M A F D R F 12 7017 65 F V A V S N P L R Y 12 7018 87 G V A S V I R G L L 12 7019 107 R L S Y C H S Q V L 12 7020 111 C H S Q V L H H S Y 12 7021 113 S Q V L H H S Y C Y 12 7022 120 Y C Y H P D V M K L 12 7023 133 D T R I N S A V G L 12 7024 137 N S A V G L T A M F 12 7025 146 F S T V G V D L L L 12 7026 148 T V G V D L L L I L 12 7027 153 L L L I L L S Y V L 12 7028 155 L I L L S Y V L I I 12 7029 173 P E E R K E T F S T 12 7030 177 K E T F S T C V S H 12 7031 183 C V S H I V A F A I 12 7032 232 V I Y S V K T K Q I 12 7033 48 F F T V M E S S V L 11 7034 68 V S N P L R Y A M I 11 7035 184 V S H I V A F A I Y 11 7036 186 H I V A F A I Y Y I 11 7037 237 K T K Q I R R A V I 11 7038 8 L S A T D L G L S I 10 7039 17 I S T L V T M L S I 10 7040 31 V R E I S F N A C L 10 7041 36 F N A C L S H M F F 10 7042 37 N A C L S H M F F I 10 7043 83 I A Q I G V A S V I 10 7044 214 V H T M I A N T Y L 10 7045 74 Y A M I L T D S R I 9 7046 127 M K L S C T D T R I 9 7047 209 Q A P A Y V H T M I 9 7048 67 A V S N P L R Y A M 8 7049 75 A M I L T D S R I A 8 7050 84 A Q I G V A S V I R 8 7051 59 A M A F D R F V A V 7 7052 61 A F D R F V A V S N 7 7053 81 S R I A Q I G V A S 7 7054 90 S V I R G L L M L T 7 7055 150 G V D L L L I L L S 7 7056 170 V A S P E E R K E T 7 7057 196 P L I S L S I V H R 7 7058 208 K Q A P A Y V H T M 7 7059 244 A V I K I L H S K E 7 7060 2 Y Y F L S M L S A T 6 7061 10 A T D L G L S I S T 6 7062 76 M I L T D S R I A Q 6 7063 121 C Y H P D V M K L S 6 7064 130 S C T D T R I N S A 6 7065 134 T R I N S A V G L T 6 7066 162 L I I R T V L S V A 6 7067 212 A Y V H T M I A N T 6 7068 235 S V K T K Q I R R A 6 7069 16 S I S T L V T M L S 5 7070 30 N V R E I S F N A C 5 7071 38 A C L S H M F F I K 5 7072 44 F F I K F F T V M E 5 7073 60 M A F D R F V A V S 5 7074 70 N P L R Y A M I L T 5 7075 71 P L R Y A M I L T D 5 7076 100 P M V A L L I R L S 5 7077 157 L L S Y V L I I R T 5 7078 158 L S Y V L I I R T V 5 7079 161 V L I I R T V L S V 5 7080 190 F A I Y Y I P L I S 5 7081 200 L S I V H R F G K Q 5 7082 227 P L M N P V I Y S V 5 7083 230 N P V I Y S V K T K 5 7084 5 L S M L S A T D L G 4 7085 13 L G L S I S T L V T 4 7086 14 G L S I S T L V T M 4 7087 23 M L S I F W F N V R 4 7088 24 L S I F W F N V R E 4 7089 33 E I S F N A C L S H 4 7090 34 I S F N A C L S H M 4 7091 42 H M F F I K F F T V 4 7092 43 M F F I K F F T V M 4 7093 47 K F F T V M E S S V 4 7094 50 T V M E S S V L L A 4 7095 51 V M E S S V L L A M 4 7096 55 S V L L A M A F D R 4 7097 80 D S R I A Q I G V A 4 7098 103 A L L I R L S Y C H 4 7099 104 L L I R L S Y C H S 4 7100 128 K L S C T D T R I N 4 7101 131 C T D T R I N S A V 4 7102 136 I N S A V G L T A M 4 7103 139 A V G L T A M F S T 4 7104 140 V G L T A M F S T V 4 7105 160 Y V L I I R T V L S 4 7106 163 I I R T V L S V A S 4 7107 192 I Y Y I P L I S L S 4 7108 201 S I V H R F G K Q A 4 7109 210 A P A Y V H T M I A 4 7110 221 T Y L L I S P L M N 4 7111 223 L L I S P L M N P V 4 7112 226 S P L M N P V I Y S 4 7113 228 L M N P V I Y S V K 4 7114 231 P V I Y S V K T K Q 4 7115 236 V K T K Q I R R A V 4 7116 242 R R A V I K I L H S 4 7117 7 M L S A T D L G L S 3 7118 9 S A T D L G L S I S 3 7119 21 V T M L S I F W F N 3 7120 28 W F N V R E I S F N 3 7121 58 L A M A F D R F V A 3 7122 79 T D S R I A Q I G V 3 7123 88 V A S V I R G L L M 3 7124 91 V I R G L L M L T P 3 7125 93 R G L L M L T P M V 3 7126 94 G L L M L T P M V A 3 7127 98 L T P M V A L L I R 3 7128 102 V A L L I R L S Y C 3 7129 109 S Y C H S Q V L H H 3 7130 122 Y H P D V M K L S C 3 7131 126 V M K L S C T D T R 3 7132 129 L S C T D T R I N S 3 7133 138 S A V G L T A M F S 3 7134 143 T A M F S T V G V D 3 7135 152 D L L L I L L S Y V 3 7136 156 I L L S Y V L I I R 3 7137 169 S V A S P E E R K E 3 7138 176 R K E T F S T C V S 3 7139 179 T F S T C V S H I V 3 7140 19 Y I P L I S L S I V 3 7141 195 I P L I S L S I V H 3 7142 199 S L S I V H R F G K 3 7143 202 I V H R F G K Q A P 3 7144 211 P A Y V H T M I A N 3 7145 21 M I A N T Y L L I S 3 7146 218 I A N T Y L L I S P 3 7147 220 N T Y L L I S P L M 3 7148 229 M N P V I Y S V K T 3 7149 3 Y F L S M L S A T D 2 7150 12 D L G L S I S T L V 2 7151 41 S H M F F I K F F T 2 7152 46 I K F F T V M E S S 2 7153 54 S S V L L A M A F D 2 7154 66 V A V S N P L R Y A 2 7155 78 L T D S R I A Q I G 2 7156 85 Q I G V A S V I R G 2 7157 92 I R G L L M L T P M 2 7158 105 L I R L S Y C H S Q 2 7159 106 I R L S Y C H S Q V 2 7160 108 L S Y C H S Q V L H 2 7161 110 Y C H S Q V L H H S 2 7162 118 H S Y C Y H P D V M 2 7163 119 S Y C Y H P D V M K 2 7164 123 H P D V M K L S C T 2 7165 132 T D T R I N S A V G 2 7166 135 R I N S A V G L T A 2 7167 142 L T A M F S T V G V 2 7168 165 R T V L S V A S P E 2 7169 167 V L S V A S P E E R 2 7170 175 E R K E T F S T C V 2 7171 180 F S T C V S G I V A 2 7172 198 I S L S I V H R F G 2 7173 206 F G K Q A P A Y V G 2 7174 222 Y L L I S P L M N P 2 7175 238 T K Q I R R A V I K 2 7176 241 I R R A V I K I L H 2 7177 243 R A V I K I L H S K 2 7178 245 V I K I L H S K E T 2 7179 1 M Y Y F L S M L S A 1 7180 22 T M L S I F W F N V 1 7181 26 I F W F N V R E I S 1 7182 29 F N V R E I S F N A 1 7183 45 F I K F F T V M E S 1 7184 57 L L A M A F D R F V 1 7185 62 F D R F V A V S N P 1 7186 64 R F V A V S N P L R 1 7187 72 L R Y A M I L T D S 1 7188 73 R Y A M I L T D S R 1 7189 82 R I A Q I G V A S V 1 7190 114 Q V L H H S Y C Y H 1 7191 116 L H H S Y C Y H P D 1 7192 117 H H S Y C Y H P D V 1 7193 124 P D V M K S C T D 1 7194

TABLE XIXB part 2 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID HLA-A*0201 10-mers v.1B: 238P1B2 58 S L H E P M Y Y F L 25 7195 8 N I T S T S I I F L 21 7196 43 L L G N S L I L F A 21 7197 5 T L Q N I T S T S I 20 7198 9 I T S T S I I F L L 20 7199 15 I F L L T G V P G L 20 7200 17 L L T G V P G L E A 20 7201 42 A L L G N S L I L F 20 7202 31 I S I P F C F L S V 19 7203 39 S V T A L L G N S L 18 7204 12 T S I I F L L T G V 17 7205 32 S I P F C F L S V T 17 7206 37 F L S V T A L L G N 17 7207 41 T A L L G N S L I L 17 7208 50 L F A T I T Q P S L 17 7209 47 S L I L F A T I T Q 16 7210 40 V T A L L G N S L T 15 7211 48 L I L F A T I T Q P 15 7212 35 F C F L S V T A L L 14 7213 53 T I T Q P S L H E P 14 7214 1 F I T S T L Q N I T 13 7215 11 S T S I I F L L T G 13 7216 14 I I F L L T G V P G 13 7217 16 F L L T G V P G L E 13 7218 24 L E A F H T W I S I 13 7219 45 G N S L I L F A T I 13 7220 49 I L F A T I T Q P S 13 7221 29 T W I S I P F C F L 12 7222 44 L G N S L I L F A T 12 7223 54 I T Q P S L H E P M 12 7224 6 L Q N I T S T S I T 11 7225 13 S I T F L L T G V P 11 7226 18 L T G V P G L E A F 11 7227 20 G V P G L E A F H T 11 7228 33 I P F C F L S V T A 11 7229 34 P F C F L S V T A L 11 7230 61 E P M Y Y F L S M L 11 7231 4 S T L Q N I T S T S 10 7232 23 G L E A F H T W I S 10 7233 3 T S T L Q N I T S T 9 7234 22 P G L E A F H T W I 9 7235 52 A T I T Q P S L H E 9 7236 2 I T S T L Q N I T S 8 7237 30 W I S I P F C F L S 8 7238 60 H E P M Y Y F L S M 7 7239 25 E A F H T W I S I P 6 7240 38 L S V T A L L G N S 6 7241 46 N S L I L F A T I T 6 7242 27 F H T W I S I P F C 5 7243 28 H T W I S I P F C F 5 7244 62 P M Y Y F L S M L S 5 7245 10 T S T S I I F L L T 4 7246 19 T G V P G L E A F H 4 7247 21 V P G L E A F H T W 4 7248 51 F A T I T Q P S L H 4 7249 36 C F L S V T A L L G 3 7250 59 L H E P M Y Y F L S 3 7251 7 Q N I T S T S I I F 2 7252 26 A F H T W I S I P F 2 7253 55 T Q P S L H E P M Y 1 7254 HLA-A*0202 10-mers v.1B: 238P1B2 24 L E A F H T W I S I 3 7255 40 V T A L L G N S L I 3 7256 50 L F A T I T Q P S L 3 7257 25 E A F H T W I S I P 2 7258 41 T A L L G N S L I L 2 7259 51 F A T I T Q P S L H 2 7260 26 A F H T W I S I P F 1 7261 42 A L L G N S L I L F 1 7262 52 A T I T Q P S L H E 1 7263 HLA-A*0203 10-mers v.1B: 238P1B2 17 L L T G V P G L E A 10 7264 33 I P F C F L S V T A 10 7265 43 L L G N S L I L F A 10 7266 18 L T G V P G L E A F 9 7267 34 P F C F L S V T A L 9 7268 44 L G N S L I L F A T 9 7269 19 T G V P G L E A F H 8 7270 35 F C F L S V T A L L 8 7271 45 G N S L I L F A T I 8 7272 HLA-A1 10-mers v.1B: 238P1B2 31 I S I P F C F L S V 17 7273 55 T Q P S L H E P M Y 16 7274 59 L H E P M Y Y F L S 16 7275 10 T S T S I I F L L T 15 7276 56 Q P S L H E P M Y Y 15 7277 11 S T S I I F L L T G 14 7278 52 A T I T Q P S L H E 13 7279 2 I T S T L Q N I T S 11 7280 23 G L E A F H T W I S 11 7281 40 V T A L L G N S L I 10 7282 54 I T Q P S L H E P M 10 7283 17 L L T G V P G L E A 9 7284 42 A L L G N S L I L F 9 7285 60 H E P M Y Y F L S M 9 7286 4 S T L Q N I T S T S 8 7287 9 I T S T S I I F L L 8 7288 36 C F L S V T A L L G 8 7289 37 F L S V T A L L G N 8 7290 43 L L G N S L I L F A 7 7291 47 S L I L F A T I T Q 7 7292 18 L T G V P G L E A F 6 7293 28 H T W I S I P F C F 6 7294 41 T A L L G N S L I L 6 7295 26 A F H T W I S I P F 5 7296 3 T S T L Q N I T S T 4 7297 7 Q N I T S T S I I F 4 7298 8 N I T S T S I I F L 4 7299 12 T S I I F L L T G V 4 7300 16 F L L T G V P G L E 4 7301 19 T G V P G L E A F H 4 7302 21 V P G L E A F H T W 4 7303 24 L E A F H T W I S I 4 7304 38 L S V T A L L G N S 4 7305 46 N S L I L F A T I T 4 7306 57 P S L H E P M Y Y F 4 7307 13 S I I F L L T G V P 3 7308 29 T W I S I P F C F L 3 7309 32 S I P F C F L S V T 3 7310 35 F C F L S V T A L L 3 7311 39 S V T A L L G N S L 3 7312 45 G N S L I L F A T I 3 7313 51 F A T I T Q P S L H 3 7314 58 S L H E P M Y Y F L 3 7315 30 W I S I P F C F L S 2 7316 62 P M Y Y F L S M L S 2 7317 1 F I T S T L Q N I T 1 7318 5 T L Q N I T S T S I 1 7319 14 I I F L L T G V P G 1 7320 15 I F L L T G V P G L 1 7321 20 G V P G L E A F H T 1 7322 27 F H T W I S I P F C 1 7323 34 P F C F L S V T A L 1 7324 49 I L F A T I T Q P S 1 7325 61 E P M Y Y F L S M L 1 7326 HLA-A26 10-mers v.1B: 238P1B2 18 L T G V P G L E A F 25 7327 42 A L L G N S L I L F 24 7328 8 N I T S T S I I F L 23 7329 9 I T S T S I I F L L 22 7330 58 S L H E P M Y Y F L 22 7331 15 I F L L T G V P G L 20 7332 28 H T W T S I P F C F 20 7333 54 I T Q P S L H E P M 20 7334 61 E P M Y Y F L S M L 20 733S 34 P F C F L S V T A L 19 7336 39 S V T A L L G N S L 19 7337 50 L F A T I T Q P S L 17 7338 26 A F H T W I S I P F 16 7339 32 S I P F C F L S V T 16 7340 53 T I T Q P S L H E P 16 7341 29 T W I S I P F C F L 15 7342 48 L I L F A T I T Q P 15 7343 11 S T S I I F L L T G 14 7344 20 G V P G L E A F H T 14 7345 55 T Q P S L H E P M Y 14 7346 57 P S L H E P M Y Y F 14 7347 60 H E P M Y Y F L S M 14 7348 14 I I F L L T G V P G 13 7349 25 E A F H T W I S I P 13 7350 37 F L S V T A L L G N 13 7351 52 A T I T Q P S L H E 13 7352 4 S T L Q N I T S T S 12 7353 7 Q N I T S T S I I F 12 7354 13 S I I F L L T G V P 12 7355 40 V T A L L G N S L I 12 7356 43 L L G N S L I L F A 12 7357 1 F I T S T L Q N I T 11 7358 17 L L T G V P G L E A 11 7359 35 F C F L S V T A L L 11 7360 49 I L F A T I T Q P S 11 7361 56 Q P S L H E P M Y Y 11 7362 2 I T S T L Q N I T S 10 7363 30 W I S I P F C F L S 10 7364 47 S L I L F A T I T Q 10 7365 5 T L Q N I T S T S I 9 7366 31 I S I P F C F L S V 9 7367 16 F L L T G V P G L E 8 7368 23 G L E A F H T W I S 8 7369 41 T A L L G N S L I L 8 7370 12 T S I I F L L T G V 7 7371 36 C F L S V T A L L G 7 7372 44 L G N S L I L F A T 6 7373 59 L H E P M Y Y F L S 6 7374 3 T S T L Q N I T S T 5 7375 10 T S T S I I F L L T 5 7376 27 F H T W I S I P F C 5 7377 38 L S V T A L L G N S 5 7378 45 G N S L I L F A T I 5 8903 21 V P G L E A F H T W 4 8904 19 T G V P G L E A F H 3 8905 33 I P F C F L S V T A 3 8906 22 P G L E A F H T W I 2 8907 2 L E A F H T W I S I 2 8908 6 L Q N I T S T S I I 1 8909 51 F A T I T Q P S L H 1 8910 62 P M Y Y F L S M L S 1 8911 HLA-A3 10-mers v.1B: 238P1B2 42 A L L G N S L I L F 21 7379 47 S L I L F A T I T Q 19 7380 14 I I F L L T G V P G 18 7381 17 L L T G V P G L E A 18 7382 39 S V T A L L G N S L 18 7383 13 S I I F L L T G V P 17 7384 32 S I P F C F L S V T 16 7385 20 G V P G L E A F H T 15 7386 5 T L Q N I T S T S I 14 7387 16 F L L T G V P G L E 14 7388 43 L L G N S L I L F A 14 7389 48 L I L F A T I T Q P 14 7390 49 I L F A T I T Q P S 14 7391 31 I S I P F C F L S V 13 7392 37 F L S V T A L L G N 13 7393 23 G L E A F H T W I S 12 7394 58 S L H E P M Y Y F L 12 7395 56 Q P S L H S P M Y Y 11 7396 4 S T L Q N I T S T S 10 7397 19 T G V P G L E A F H 10 7398 33 I P F C F L S V T A 10 7399 7 Q N I T S T S I I F 9 7400 11 S T S I I F L L T G 9 7401 30 W I S I P F C F L S 9 7402 51 F A T I T Q P S L H 9 7403 52 A T I T Q P S L H E 9 7404 1 F I T S T L Q N I T 8 7405 8 N I T S T S I I F L 8 7406 36 C F L S V T A L L G 8 7407 45 G N S L I L F A T I 8 7408 53 T I T Q P S L H E P 8 7409 26 A F H T W I S I P F 7 7410 46 N S L I L F A T I T 7 7411 55 T Q P S L H E P M Y 7 7412 2 I T S T L Q N I T S 6 7413 28 H T W I S I P F C F 6 7414 41 T A L L G N S L I L 6 7415 57 P S L H E P M Y Y F 6 7416 12 T S I I F L L T G V 5 7417 15 I F L L T G V P G L 5 7418 18 L T G V P G L E A F 5 7419 21 V P G L E A F H T W 5 7420 22 P G L E A F H T W I 5 7421 40 V T A L L G N S L I 5 7422 54 I T Q P S L H E P M 5 7423 60 H E P M Y Y F L S M 5 7424 62 P M Y Y F L S M L S 5 7425 3 T S T L Q N I T S T 4 7426 6 L Q N I T S T S I I 4 7427 35 F C F L S V T A L L 4 7428 61 E P M Y Y F L S M L 4 7429 10 T S T S I I F L L T 3 7430 2 L E A F H T W I S I 3 7431 25 E A F H T W I S I P 3 7432 29 T W I S I P F C F L 3 7433 50 L F A T I T Q P S L 3 7434 59 L H E P M Y Y F L S 3 7435 9 I T S T S I T F L L 2 7436 34 P F C F L S V T A L 2 7437 38 L S V T A L L G N S 1 7438 44 L G N S L I L F A T 1 7439 HLA-B*0702 10-mers v.1B: 238P1B2 61 E P M Y Y F L S M L 22 7440 33 I P F C F L S V T A 19 7441 9 I T S T S I I F L L 13 7442 15 I F L L T G V P G L 13 7443 34 P F C F L S V T A L 13 7444 21 V P G L E A F H T W 12 7445 29 T W I S I P F C F L 12 7446 35 F C F L S V T A L L 12 7447 41 T A L L G N S L I L 12 7448 50 L F A T I T Q P S L 12 7449 56 Q P S L H E P M Y Y 12 7450 8 N I T S T S I T F L 11 7451 31 I S T P F C F L S V 11 7452 39 S V T A L L G N S L 11 7453 58 S L H E P M Y Y F L 11 7454 17 L L T G V P G L E A 10 7455 42 A L L G N S L I L F 10 7456 43 L L G N S L I L F A 10 7457 45 G N S L I L F A T I 10 7458 10 T S T S I I F L L T 9 7459 26 A F H T W I S I P F 9 7460 54 I T Q P S L H E P M 9 7461 60 H E P M Y Y F L S M 9 7462 18 L T G V P G L E A F 8 7463 20 G V P G L E A F H T 8 7464 3 T S T L Q N I T S T 7 7465 5 T L Q N I T S T S I 7 7466 6 L Q N I T S T S I I 7 7467 12 T S T I F L L T G V 7 7468 22 P G L E A F H T W I 7 7469 24 L E A F H T W I S I 7 7470 32 S I P F C F L S V T 7 7471 40 V T A L L G N S L I 7 7472 44 L G N S L I L F A T 7 7473 46 N S L I L F A T I T 7 7474 1 F I T S T L Q N I T 6 7475 7 Q N I T S T S I I F 6 7476 28 H T W I S I P F C F 6 7477 57 P S L H E P M Y Y F 6 7478 11 S T S T T F L L T G 4 7479 14 I I F L L T G V P G 4 7480 37 F L S V T A L L G N 4 7481 49 I L F A T I T Q P S 4 7482 52 A T I T Q P S L H E 4 7483 2 I T S T L Q N I T S 3 7484 19 T G V P G L E A F H 3 7485 13 S I I F L L T G V P 2 7486 23 G L E A F H T W I S 2 7487 27 F H T W I S I P F C 2 7488 30 W I S I P F C F L S 2 7489 36 C F L S V T A L L G 2 7490 4 S T L Q N I T S T S 1 7491 25 E A F H T W I S I P 1 7492 47 S L I L F A T I T Q 1 7493 48 L I L F A T I T Q P 1 7494 55 T Q P S L H E P M Y 1 7495 59 L H E P M Y Y F L S 1 7496 HLA-B*4402 10-mers v.1B: 238P1B2 24 L E A F H T W I S I 19 7497 42 A L L G N S L I L F 18 7498 9 I T S T S I I F L L 16 7499 7 Q N I T S T S I I F 15 7500 8 N I T S T S I I F L 15 7501 26 A F H T W I S I P F 15 7502 35 F C F L S V T A L L 15 7503 21 V P G L E A F H T W 14 7504 29 T W I S I P F C F L 14 7505 61 E P M Y Y F L S M L 14 7506 15 I F L L T G V P G L 13 7507 18 L T G V P G L E A F 13 7508 34 P F C F L S V T A L 13 7509 41 T A L L G N S L I L 13 7510 56 Q P S L H E P M Y Y 13 7511 60 H E P M Y Y F L S M 13 7512 39 S V T A L L G N S L 12 7513 45 G N S L I L F A T I 12 7514 28 H T W I S I P F C F 11 7515 50 L F A T I T Q P S L 11 7516 55 T Q P S L H E P M Y 11 7517 57 P S L H E P M Y Y F 11 7518 58 S L H E P M Y Y F L 11 7519 40 V T A L L G N S L I 10 7520 5 T L Q N I T S T S I 9 7521 6 L Q N I T S T S I I 9 7522 22 P G L E A F H T W I 9 7523 31 I S I P F C F L S V 8 7524 47 S L I L E A T I T Q 8 7525 52 A T I T Q P S L H E 8 7526 25 E A F H T W I S I P 7 7527 13 S I I F L L T G V P 6 7528 11 S T S I I F L L T G 5 7529 43 L L G N S L I L F A 5 7530 48 L I L F A T I T Q P 5 7531 2 I T S T L Q N I T S 4 7532 3 T S T L Q N I T S T 4 7533 4 S T L Q N I T S T S 4 7534 12 T S I I F L L T G V 4 7535 14 I I F L L T G V P G 4 7536 16 F L L T G V P G L E 4 7537 32 S I P F C F L S V T 4 7538 33 I P F C F L S V T A 4 7539 36 C F L S V T A L L G 4 7540 59 L H E P M Y Y F L S 4 7541 10 T S T S I I F L L T 7542

TABLE XIXB part 3 Pos 1 2 3 4 5 6 7 8 9 0 score SEQ ID HLA-A*0201 10-mers v.2: 238P1B2 8 L L T S P L M N P V 22 7543 7 Y L L T S P L M N P 18 7544 2 M I A N T Y L L T S 17 7545 9 L T S P L M N P V I 17 7546 1 T M I A N T Y L L T 15 7547 3 I A N T Y L L T S P 13 7548 4 A N T Y L L T S P L 10 7549 5 N T Y L L T S P L M 10 7550 6 T Y L L T S P L M N 3 7551 10 T S P L M N P V I Y 2 7552 HLA-A*0202 10-mers v.2: 238P1B2 2 M I A N T Y L L T S 3 7553 3 I A N T Y L L T S P 2 7554 4 A N T Y L L T S P L 1 7555 HLA-A1 10-mers v.2: 238P1B2 10 T S P L M N P V I Y 19 7556 9 L T S P L M N P V I 12 7557 1 T M I A N T Y L L T 8 7558 5 N T Y L L T S P L M 8 7559 6 T Y L L T S P L M N 7 7560 2 M I A N T Y L L T S 6 7561 7 Y L L T S P L M N P 3 7562 4 A N T Y L L T S P L 1 7563 8 L L T S P L M N P V 1 7564 HLA-A26 10-mers v.2: 238P1B2 5 N T Y L L T S P L M 19 7565 2 M I A N T Y L L T S 15 7566 7 Y L L T S P L M N P 14 7567 8 L L T S P L M N P V 13 7568 9 L T S P L M N P V I 13 7569 10 T S P L M N P V I Y 12 7570 4 A N T Y L L T S P L 9 7571 1 T M I A N T Y L L T 6 7572 3 I A N T Y L L T S P 5 7573 6 T Y L L T S P L M N 1 7574 HLA-A3 10-mers v.2: 238P1B2 2 M I A N T Y L L T S 17 7575 7 Y L L T S P L M N P 13 7576 10 T S P L M N P V I Y 12 7577 8 L L T S P L M N P V 10 7578 1 T M I A N T Y L L T 8 7579 6 T Y L L T S P L M N 8 7580 9 L T S P L M N P V I 7 7581 4 A N T Y L L T S P L 6 7582 3 I A N T Y L L T S P 5 7583 5 N T Y L L T S P L M 5 7584 HLA-B*0702 10-mers v.2: 238P1B2 4 A N T Y L L T S P L 14 7585 9 L T S P L M N P V I 11 7586 1 T M I A N T Y L L 7587 T

TABLE XIXC part 1 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID HLA-DRB1*0101 15-mers v.1: 238P1B2 2 Y Y F L S M L S A T D L G L S 34 7588 151 V D L L L I L L S Y V L I I R 33 7589 46 I K F F T V M E S S V L L A M 32 7590 191 A I Y Y I P L I S L S I V H R 32 7591 203 V H R F G K Q A P A Y V H T M 32 7592 92 I R G L L M L T P M V A L L I 31 7593 95 L L M L T P M V A L L I R L S 31 7594 158 L S Y V L I I R T V L S V A S 31 7595 14 G L S I S T L V T M L S I F W 30 7596 161 V L I I R T V L S V A S P E E 30 7597 181 S T C V S H I V A F A I Y Y I 30 7598 230 N P V I Y S V K T K Q I R R A 29 7599 157 L L S Y V L I I R T V L S V A 27 7600 18 S T L V T M L S I F W F N V R 26 7601 89 A S V I R G L L M L T P M V A 26 7602 98 L T P M V A L L I R L S Y C H 26 7603 199 S L S I V H R F G K Q A P A Y 26 7604 33 E I S F N A C L S H M F F I K 25 7605 133 D T R I N S A V G L T A M F S 25 7606 136 I N S A V G L T A M F S T V G 25 7607 143 T A M F S T V G V D L L L I L 25 7608 148 T V G V D L L L I L L S Y V L 25 7609 152 D L L L I L L S Y V L I I R T 25 7610 10 A T D L G L S I S T L V T M L 24 7611 25 S I F W F N V R E I S F N A C 24 7612 45 F I K F F T V M E S S V L L A 24 7613 51 V M E S S V L L A M A F D R F 24 7614 73 R Y A M I L T D S R I A Q I G 24 7615 102 V A L L I R L S Y C H S Q V L 24 7616 142 L T A M F S T V Q V D L L L I 24 7617 177 K E T F S T C V S H I V A F A 24 7618 222 Y L L I S P L M N P V I Y S V 24 7619 37 N A C L S H M F F I K F F T V 23 7620 40 L S H M F F I K F F T V M E S 23 7621 71 P L R Y A M I L T D S R I A Q 23 7622 80 D S R I A Q I G V A S V I R G 23 7623 131 C T D T R I N S A V G L T A M 23 7624 150 G V D L L L I L L S Y V L I I 23 7625 192 I Y Y I P L I S L S I V H R F 23 7626 218 I A N T Y L L I S P L M N P V 23 7627 219 A N T Y L L I S P L M N P V I 23 7628 1 M Y Y F L S M L S A T D L G L 22 7629 20 L V T M L S I F W F N V R E I 22 7630 41 S H M F F I K F F T V M E S S 22 7631 54 S S V L L A M A F D R F V A V 22 7632 79 T D S R I A Q I Q V A S V I R 22 7633 85 Q I G V A S V I R G L L M L T 22 7634 86 I G V A S V I R G L L M L T P 22 7635 105 L I R L S Y C H S Q V L H H S 22 7636 139 A V G L T A M F S T V G V D L 22 7637 189 A F A I Y Y I P L I S L S I V 22 7638 211 P A Y V H T M I A N T Y L L I 22 7639 17 I S T L V T M L S I F W F N V 21 7640 91 V I R G L L M L T P M V A L L 21 7641 187 I V A F A I Y Y I P L I S L S 21 7642 190 F A I Y Y I P L I S L S I V H 21 7643 164 I R T V L S V A S P E E R K E 20 7644 208 K Q A P A Y V H T M I A N T Y 20 7645 12 D L G L S I S T L V T M L S I 19 7646 24 L S I F W F N V R E I S F N A 19 7647 72 L R Y A M I L T D S R I A Q I 19 7648 81 S R I A Q I G V A S V I R G L 19 7649 119 S Y C Y H P D V M K L S C T D 19 7650 123 H P D V M K L S C T D T R I N 19 7651 159 S Y V L I I R T V L S V A S P 19 7652 197 L I S L S I V H R F G K Q A P 19 7653 220 N T Y L L I S P L M N P V I Y 19 7654 238 T K Q I R R A V I K I L H S K 19 7655 4 F L S M L S A T D L G L S I S 18 7656 7 M L S A T D L G L S I S T L V 18 7657 42 H M F F I K F F T V M E S S V 18 7658 43 M F F I K F F T V M E S S V L 18 7659 55 S V L L A M A F D R F V A V S 18 7660 59 A M A F D R F V A V S N P L R 18 7661 90 S V I R G L L M L T P M V A L 18 7662 233 I Y S V K T K Q I R R A V I K 18 7663 234 Y S V K T K Q I R R A V I R I 18 7664 23 M L S I F W F N V R E I S F N 17 7665 53 E S S V L L A M A F D R F V A 17 7666 57 L L A M A F D R F V A V S N P 17 7667 60 M A F D R F V A V S N P L R Y 17 7668 62 F D R F V A V S N P L R Y A M 17 7669 66 V A V S N P L R Y A M I L T D 17 7670 67 A V S N P L R Y A M I L T D S 17 7671 75 A M I L T D S R I A Q I G V A 17 7672 93 R G L L M L T P M V A L L I R 17 7673 99 T P M V A L L I R L S Y C H S 17 7674 101 M V A L L I R L S Y C H S Q V 17 7675 140 V G L T A M F S T V G V D L L 17 7676 147 S T V G V D L L L I L L S Y V 17 7677 153 L L L I L L S Y V L I I R T V 17 7678 156 I L L S Y V L I I R T V L S V 17 7679 162 L I I R T V L S V A S P E E R 17 7680 184 V S H I V A F A I Y Y I P L I 17 7681 185 S H I V A F A I Y Y I P L I S 17 7682 195 I P L I S L S I V H R F G K Q 17 7683 200 L S I V H R F G K Q A P A Y V 17 7684 212 A Y V H T M I A N T Y L L I S 17 7685 213 Y V H T M I A N T Y L L I S P 17 7686 217 M I A N T Y L L I S P L M N P 17 7687 5 L S M L S A T D L G L S I S T 16 7688 6 S M L S A T D L G L S I S T L 16 7689 9 S A T D L Q L S I S T L V T M 16 7690 15 L S I S T L V T M L S I F W F 16 7691 28 W F N V R E I S F N A C L S H 16 7692 30 N V R E I S F N A C L S H M F 16 7693 34 I S F N A C L S H M F F I K F 16 7694 47 K F F T V M E S S V L L A M A 16 7695 48 F F T V M E S S V L L A M A F 16 7696 49 F T V M E S S V L L A M A F D 16 7697 65 F V A V S N P L R Y A M I L T 16 7698 77 I L T D S R T A Q I G V A S V 16 7699 94 G L L M L T P M V A L L I R L 16 7700 113 S Q V L H H S Y C Y H P D V M 16 7701 124 P D V M K L S C T D T R I N S 16 7702 134 T R I N S A V G L T A M F S T 16 7703 146 F S T V G V D L L L I L L S Y 16 7704 160 Y V L I I R T V L S V A S P E 16 7705 173 P E E R K E T F S T C V S H I 16 7706 194 Y I P L I S L S I V H R F G K 16 7707 214 V H T M I A N T Y L L I S E L 16 7708 215 H T M I A N T Y L L I S P L M 16 7709 221 T Y L L I S P L M N P V I Y S 16 7710 225 I S P L M N P V I Y S V K T K 16 7711 27 F W F N V R E I S F N A C L S 15 7712 70 N P L R Y A M I L T D S R I A 15 7713 126 V M K L S C T D T R I N S A V 15 7714 145 M F S T V G V D L L L I L L S 15 7715 178 E T F S T C V S H I V A F A I 15 7716 226 S P L M N P V I Y S V K T K Q 15 7717 235 S V K T K Q I R R A V I K I L 15 7718 50 T V M E S S V L L A M A F D R 14 7719 61 A F D R F V A V S N P L R Y A 14 7720 63 D R F V A V S N P L R Y A M I 14 7721 108 L S Y C H S Q V L H H S Y C Y 14 7722 116 L H H S Y C Y H P D V M K L S 14 7723 135 R I N S A V G L T A M F S T V 14 7724 155 L I L L S Y V L I I R T V L S 14 7725 165 R T V L S V A S P E E R K E T 14 7726 174 E E R K E T F S T C V S H I V 14 7727 186 H I V A F A I Y Y I P L I S L 14 7728 188 V A F A I Y Y I P L I S L S I 14 7729 240 Q I R R A V I K I L H S K E T 14 7730 78 L T D S R I A Q I G V A S V I 13 7731 87 G V A S V I R G L L M L T P M 13 7732 130 S C T D T R I N S A V G L T A 13 7733 223 L L I S P L M N P V I Y S V K 13 7734 227 P L M N P V I Y S V K T K Q I 13 7735 11 T D L G L S I S T L V T M L S 12 7736 26 I F W F N V R E I S F N A C L 12 7737 82 R I A Q I G V A S V I R G L L 12 7738 84 A Q I G V A S V I R G L L M L 12 7739 21 V T M L S I F W F N V R E I S 11 7740 29 F N V R E I S F N A C L S H M 11 7741 103 A L L I R L S Y C H S Q V L H 11 7742 167 V L S V A S P E E R K E T F S 11 7743 201 S I V H R F G K Q A P A Y V H 11 7744 210 A P A Y V H T M I A N T Y L L 11 7745 236 V K T K Q I R R A V I K I L H 11 7746 38 A C L S H M F F I K F F T V M 10 7747 52 M E S S V L L A M A F D R F V 10 7748 64 R F V A V S N P L R Y A M I L 10 7749 69 S N P L R Y A M I L T D S R I 10 7750 107 R L S Y C H S Q V L H H S Y C 10 7751 117 H H S Y C Y H P D V M K L S C 10 7752 163 I I R T V L S V A S P E E R K 10 7753 229 M N P V I Y S V K T K Q I R R 10 7754 231 P V I Y S V K T K Q I R R A V 10 7755 13 L G L S I S T L V T M L S I F 9 7756 31 V R E I S F N A C L S H M F F 9 7757 35 S F N A C L S H M F F I K F F 9 7758 44 F F I K F F T V M E S S V L L 9 7759 118 H S Y C Y H P D V M K L S C T 9 7760 125 D V M K L S C T D T R I N S A 9 7761 128 K L S C T D T R I N S A V G L 9 7762 137 N S A V G L T A M F S T V G V 9 7763 144 A M F S T V G V D L L L I L L 9 7764 149 V G V D L L L I L L S Y V L I 9 7765 170 V A S P E E R K E T F S T C V 9 7766 180 F S T C V S H I V A F A I Y Y 9 7767 182 T C V S H I V A F A I Y Y I P 9 7768 193 Y Y I P L I S L S I V H R F G 9 7769 204 H R F G K Q A P A Y V H T M I 9 7770 207 G K Q A P A Y V H T M I A N T 9 7771 216 T M I A N T Y L L I S P L M N 9 7772 224 L I S P L M N P V I Y S V K T 9 7773 237 K T K Q I R R A V I K I L H S 9 7774 16 S I S T L V T M L S I F W F N 8 7775 32 R E I S F N A C L S H M F F I 8 7776 56 V L L A M A F D R F V A V S N 8 7777 58 L A M A F D R F V A V S N P L 8 7778 74 Y A M I L T D S R I A Q I G V 8 7779 76 M I L T D S R I A Q I G V A S 8 7780 83 I A Q I G V A S V I R G L L M 8 7781 88 V A S V I R G L L M L T P M V 8 7782 97 M L T P M V A L L I R L S Y C 8 7783 104 L L I R L S Y C H S Q V L H H 8 7784 110 Y C H S Q V L H H S Y C Y H P 8 7785 111 C H S Q V L H H S Y C Y H P D 8 7786 112 H S Q V L H H S Y C Y H P D V 8 7787 115 V L H H S Y C Y H P D V M K L 8 7788 122 Y H P D V M K L S C T D T R I 8 7789 127 M K L S C T D T R I N S A V G 8 7790 129 L S C T D T R I N S A V G L T 8 7791 138 S A V G L T A M F S T V G V D 8 7792 154 L L I L L S Y V L I I R T V L 8 7793 169 S V A S P E E R K E T F S T C 8 7794 176 R K E T F S T C V S H I V A F 8 7795 179 T F S T C V S H I V A F A I Y 8 7796 183 C V S H I V A F A I Y Y I P L 8 7797 202 I V H R F G K Q A P A Y V H T 8 7798 206 F G K Q A P A Y V H T M I A N 8 7799 239 K Q I R R A V I K I L H S K E 8 7800 3 Y F L S M L S A T D L G L S I 7 7801 8 L S A T D L G L S I S T L V T 7 7802 96 L M L T P M V A L L I R L S Y 7 7803 100 P M V A L L I R L S Y C H S Q 7 7804 171 A S P E E R K E T F S T C V S 7 7805 196 P L I S L S I V H R F G K Q A 7 7806 109 S Y C H S Q V L H H S Y C Y H 6 7807 120 Y C Y H P D V M K L S C T D T 6 7808 121 C Y H P D V M K L S C T D T R 6 7809 228 L M N P V I Y S V K T K Q I R 5 7810 172 S F E E R K E T F S T C V S H 4 7811 232 V I Y S V K T K Q I R R A V I 3 7812 168 L S V A S P E E R K E T F S T 2 7813 175 E R K E T F S T C V S H I V A 2 7814 19 T L V T M L S I F W F N V R E 1 7815 22 T M L S I F W F N V R E I S F 1 7816 39 C L S H M F F I K F F T V M E 1 7817 141 G L T A M F S T V G V D L L L 1 7818 166 T V L S V A S P E E R K E T F 1 7819 198 I S L S I V H R F G K Q A P A 1 7820 HLA-DRB1*0301 (DR17) 15-mers v.1: 238P1B2 74 Y A M I L T D S R I A Q I G V 30 7821 146 F S T V G V D L L L I L L S Y 30 7822 85 Q I G V A S V I R G L L M L T 26 7823 99 T P M V A L L I R L S Y C H S 26 7824 4 F L S M L S A T D L G L S I S 22 7825 57 L L A M A F D R F V A V S N F 22 7826 93 R G L L M L T P M V A L L I R 22 7827 94 G L L M L T P M V A L L I R L 22 7828 18 S T L V T M L S I F W F N V R 21 7829 151 V D L L L I L L S Y V L I I R 21 7830 2 Y Y F L S M L S A T D L G L S 20 7831 54 S S V L L A M A F D R F V A V 20 7832 142 L T A M F S T V G V D L L L I 20 7833 37 N A C L S H M F F I K F F T V 19 7834 63 D R F V A V S N P L R Y A M I 19 7835 105 L I R L S Y C H S Q V L H H S 19 7836 167 V L S V A S P E E R K E T F S 19 7837 189 A F A I Y Y I P L I S L S I V 19 7838 195 I P L I S L S I V H R F G K Q 19 7839 200 L S I V H R F G K Q A P A Y V 19 7840 211 P A Y V H T M I A N T Y L L I 19 7841 238 T K Q I R R A V I K I L H S K 19 7842 46 I K F F T V M E S S V L L A M 18 7843 65 F V A V S N P L R Y A M I L T 18 7844 143 T A M F S T V G V D L L L I L 18 7845 229 M N P V I Y S V K T K Q I R R 18 7846 33 E I S F N A C L S H M F F I K 17 7847 62 F D R F V A V S N P L R Y A M 17 7848 127 M K L S C T D T R I N S A V G 17 7849 222 Y L L I S P L M N P V I Y S V 17 7850 157 L L S Y V L I I R T V L S V A 16 7851 197 L I S L S I V H R F G K Q A P 16 7852 212 A Y V H T M I A N T Y L L I S 16 7853 28 W F N V R E I S F N A C L S H 15 7854 29 F N V R E I S F N A C L S H M 15 7855 152 D L L L I L L S Y V L I I R T 15 7856 169 S V A S P E E R K E T F S T C 15 7857 20 L V T M L S I F W F N V R E I 14 7858 24 L S I F W F N V R E I S F N A 14 7859 25 S I F W F N V R E I S F N A C 14 7860 45 F I K F F T V M E S S V L L A 14 7861 48 F F T V M E S S V L L A M A F 14 7862 95 L L M L T P M V A L L I R L S 14 7863 148 T V G V D L L L I L L S Y V L 14 7864 150 G V D L L L I L L S Y V L I I 14 7865 153 L L L I L L S Y V L I I R T V 14 7866 158 L S Y V L I I R T V L S V A S 14 7867 161 V L I I R T V L S V A S P E E 14 7868 194 Y I P L I S L S I V H R F G K 14 7869 220 N T Y L L I S P L M N P V I Y 14 7870 221 T Y L L I S P L M N P V I Y S 14 7871 1 M Y Y F L S M L S A T D L G L 13 7872 47 K F F T V M E S S V L L A M A 13 7873 49 F T V M E S S V L L A M A F D 13 7874 53 E S S V L L A M A F D R F V A 13 7875 88 V A S V I R G L L M L T P M V 13 7876 89 A S V I R Q L L M L T P M V A 13 7877 92 I R G L L M L T P M V A L L I 13 7878 98 L T P M V A L L I R L S Y C H 13 7879 101 M V A L L I R L S Y C H S Q V 13 7880 112 H S Q V L H H S Y C Y H P D V 13 7881 154 L L I L L S Y V L I I R T V L 13 7882 164 I R T V L S V A S P E E R K E 13 7883 214 V H T M I A N T Y L L I S P L 13 7884 6 S M L S A T D L G L S I S T L 12 7885 10 A T D L G L S I S T L V T M L 12 7886 17 I S T L V T M L S I F W F N V 12 7887 69 S N P L R Y A M I L T D S R I 12 7888 73 R Y A M I L T D S R I A Q I G 12 7889 75 A M I L T D S R I A Q I G V A 12 7890 80 D S R I A Q I G V A S V I R G 12 7891 102 V A L L I R L S Y C H S Q V L 12 7892 103 A L L I R L S Y C H S Q V L H 12 7893 119 S Y C Y H P D V M K L S C T D 12 7894 124 P D V M K L S C T D T R I N S 12 7895 133 D T R I N S A V G L T A M F S 12 7896 149 V G V D L L L I L L S Y V L I 12 7897 159 S Y V L I I R T V L S V A S P 12 7898 192 I Y Y I P L I S L S I V H R F 12 7899 230 N P V I Y S V K T K Q I R R A 12 7900 5 L S M L S A T D L G L S I S T 11 7901 9 S A T D L G L S I S T L V T M 11 7902 12 D L G L S I S T L V T M L S I 11 7903 13 L G L S I S T L V T M L S I F 11 7904 14 G L S I S T L V T M L S I F W 11 7905 21 V T M L S I F W F N V R E I S 11 7906 31 V R E I S F N A C L S H M F F 11 7907 40 L S H M F F I K F F T V M E S 11 7908 43 M F F I K F F T V M E S S V L 11 7909 55 S V L L A M A F D R F V A V S 11 7910 83 I A Q I G V A S V I R G L L M 11 7911 87 G V A S V I R G L L M L T P M 11 7912 123 H P D V M K L S C T D T R I N 11 7913 131 C T D T R I N S A V G L T A M 11 7914 137 N S A V G L T A M F S T V G V 11 7915 139 A V G L T A M F S T V G V D L 11 7916 144 A M F S T V G V D L L L I L L 11 7917 147 S T V G V D L L L I L L S Y V 11 7918 155 L I L L S Y V L I I R T V L S 11 7919 160 Y V L I I R T V L S V A S P E 11 7920 165 R T V L S V A S P E E R K E T 11 7921 181 S T C V S H I V A F A I Y Y I 11 7922 184 V S H I V A F A I Y Y I P L I 11 7923 199 S L S I V H R F G K Q A P A Y 11 7924 215 H T M I A N T Y L L I S P L M 11 7925 226 S P L M N P V I Y S V K T K Q 11 7926 233 I Y S V K T K Q I R R A V I K 11 7927 16 S I S T L V T M L S I F W F N 10 7928 23 M L S I F W F N V R E I S F N 10 7929 42 H M F F I K F F T V M E S S V 10 7930 51 V M E S S V L L A M A F D R F 10 7931 111 C H S Q V L H H S Y C Y H P D 10 7932 113 S Q V L H H S Y C Y H P D V M 10 7933 126 V M K L S C T D T R I N S A V 10 7934 135 R I N S A V G L T A M F S T V 10 7935 179 T F S T C V S H I V A F A I Y 10 7936 185 S H I V A F A I Y Y I P L I S 10 7937 217 M I A N T Y L L I S P L M N P 10 7938 223 L L I S P L M N P V I Y S V K 10 7939 225 I S P L M N P V I Y S V K T K 10 7940 26 I F W F N V R E I S F N A C L 9 7941 34 I S F N A C L S H M F F I K F 9 7942 38 A C L S H M F F I K F F T V M 9 7943 59 A M A F D R F V A V S N P L R 9 7944 61 A F D R F V A V S N P L R Y A 9 7945 67 A V S N P L R Y A M I L T D S 9 7946 84 A Q I G V A S V I R G L L M L 9 7947 97 M L T P M V A L L I R L S Y G 9 7948 106 I R L S Y C H S Q V L H H S Y 9 7949 109 S Y C H S Q V L H H S Y C Y H 9 7950 118 H S Y C Y H P D V M K L S C T 9 7951 182 T C V S H I V A F A I Y Y I P 9 7952 183 C V S H I V A F A I Y Y I P L 9 7953 187 I V A F A I Y Y I P L I S L S 9 7954 201 S I V H R F G K Q A P A Y V H 9 7955 202 I V H R F G K Q A P A Y V H T 9 7956 203 V H R F C K Q A P A Y V H T M 9 7957 213 Y V H T M I A N T Y L L I S P 9 7958 22 T M L S I F W F N V R E I S F 8 7959 41 S H M F F I K F F T V M E S S 8 7960 166 T V L S V A S P E E R K E T F 8 7961 168 L S V A S P E E R K E T F S T 8 7962 170 V A S P E E R K E T F S T C V 8 7963 177 K E T F S T C V S H I V A F A 8 7964 186 H I V A F A I Y Y I P L I S L 8 7965 232 V I Y S V K T K Q I R R A V I 8 7966 234 Y S V K T K Q I R R A V I K I 8 7967 235 S V K T K Q I R R A V I K I L 8 7968 239 K Q I R R A V T K I L H S K E 8 7969 120 Y C Y H P D V M K L S C T D T 7 7970 129 L S C T D T R I N S A V G L T 7 7971 231 P V I Y S V K T K Q I R R A V 7 7972 39 C L S H M F F I K F F T V M E 6 7973 56 V L L A M A F D R F V A V S N 6 7974 77 I L T D S R I A Q I G V A S V 6 7975 11 T D L G L S I S T L V T M L S 5 7976 219 A N T Y L L I S P L M N P V I 5 7977 91 V I R G L L M L T P M V A L L 4 7978 224 L I S P L M N P V I Y S V K T 4 7979 15 L S I S T L V T M L S I F W F 3 7980 30 N V R E I S F N A C L S H M F 3 7981 36 F N A C L S H M F F I K F F T 3 7982 50 T V M E S S V L L A M A F D R 3 7983 52 M E S S V L L A M A F D R F V 3 7984 64 R F V A V S N P L R Y A M I L 3 7985 68 V S N P L R Y A M I L T D S R 3 7986 79 T D S R I A Q I G V A S V I R 3 7987 100 P M V A L L I R L S Y C H S Q 3 7988 104 L L I R L S Y C H S Q V L H H 3 7989 122 Y H P D V M K L S C T D T R I 3 7990 125 D V M K L S C T D T R I N S A 3 7991 140 V G L T A M F S T V G V D L L 3 7992 145 M F S T V G V D L L L I L L S 3 7993 162 L I I R T V L S V A S P E E R 3 7994 180 F S T C V S H I V A F A I Y Y 3 7995 191 A I Y Y I P L I S L S I V H R 3 7996 193 Y Y I P L I S L S I V H R F G 3 7997 196 P L I S L S I V H R F G K Q A 3 7998 210 A P A Y V H T M I A N T Y L L 3 7999 3 Y F L S M L S A T D L G L S I 2 8000 7 M L S A T D L G L S I S T L V 2 8001 8 L S A T D L G L S I S T L V T 2 8002 27 F W F N V R E I S F N A C L S 2 8003 60 M A F D R F V A V S N P L R Y 2 8004 66 V A V S N P L R Y A M I L T D 2 8005 71 P L R Y A M I L T D S R I A Q 2 8006 76 M I L T D S R I A Q I G V A S 2 8007 82 R I A Q I G V A S V I R G L L 2 8008 86 I G V A S V I R G L L M L T P 2 8009 96 L M L T P M V A L L I R L S Y 2 8010 117 H H S Y C Y H P D V M K L S C 2 8011 128 K L S C T D T R I N S A V G L 2 8012 130 S C T D T R I N S A V G L T A 2 8013 132 T D T R I N S A V G L T A M F 2 8014 134 T R I N S A V G L T A M F S T 2 8015 138 S A V G L T A M F S T V G V D 2 8016 163 I I R T V L S V A S P E E R K 2 8017 173 P E E R K E T F S T C V S H I 2 8018 175 E R K E T F S T C V S H I V A 2 8019 178 E T F S T C V S H I V A F A I 2 8020 188 V A F A I Y Y I P L I S L S I 2 8021 190 F A I Y Y I P L I S L S I V H 2 8022 198 I S L S I V H R F G K Q A P A 2 8023 204 H R F G K Q A P A Y V H T M I 2 8024 218 I A N T Y L L I S P L M N P V 2 8025 227 P L M N P V I Y S V K T K Q I 2 8026 228 L M N P V I Y S V K T K Q I R 2 8027 237 K T K Q I R R A V I K I L H S 2 8028 240 Q I R R A V I K I L H S K E T 2 8029 19 T L V T M L S I F W F N V R E 1 8030 35 S F N A C L S H M F F I K F F 1 8031 58 L A M A F D R F V A V S N P L 1 8032 78 L T D S R I A Q I G V A S V I 1 8033 90 S V I R G L L M L T P M V A L 1 8034 107 R L S Y C H S Q V L H H S Y C 1 8035 108 L S Y C H S Q V L H H S Y C Y 1 8036 114 Q V L H H S Y C Y H P D V M K 1 8037 121 C Y H P D V M K L S C T D T R 1 8038 136 I N S A V G L T A M F S T V G 1 8039 171 A S P E E R K E T F S T C V S 1 8040 172 S P E E R K E T F S T C V S H 1 8041 174 E E R K E T F S T C V S H I V 1 8042 176 R K E T F S T C V S H I V A F 1 8043 205 R F G K Q A P A Y V H T M I A 1 8044 206 F G K Q A P A Y V H T M I A N 1 8045 209 Q A P A Y V H T M I A N T Y L 1 8046 216 T M I A N T Y L L I S P L M N 1 8047 236 V K T K Q I R R A V I K I L H 1 8048 HLA-DRB1*0401 (DR4Dw4) 15-mers v.1: 238P1B2 157 L L S Y V L I I R T V L S V A 28 8049 191 A I Y Y I P L I S L S I V H R 28 8050 14 G L S I S T L V T M L S I F W 26 8051 74 Y A M I L T D S R I A Q I G V 26 8052 85 Q I G V A S V I R G L L M L T 26 8053 151 V D L L L I L L S Y V L I I R 26 8054 158 L S Y V L I I R T V L S V A S 26 8055 161 V L I I R T V L S V A S P E E 26 8056 197 L I S L S I V H R F G K Q A P 26 8057 222 Y L L I S P L M N P V I Y S V 26 8058 230 N P V I Y S V K T K Q I R R A 26 8059 24 L S I F W F N V R E I S F N A 22 8060 42 H M F F I K F F T V M E S S V 22 8061 45 F I K F F T V M E S S V L L A 22 8062 46 I K F F T V M E S S V L L A M 22 8063 59 A M A F D R F V A V S N P L R 22 8064 62 F D R F V A V S N P L R Y A M 22 8065 71 P L R Y A M I L T D S R I A Q 22 8066 119 S Y C Y H P D V M K L S C T D 22 8067 143 T A M F S T V G V D L L L I L 22 8068 177 K E T F S T C V S H I V A F A 22 8069 219 A N T Y L L I S P L M N P V I 22 8070 2 Y Y F L S M L S A T D L G L S 20 8071 4 F L S M L S A T D L G L S I S 20 8072 10 A T D L G L S I S T L V T M L 20 8073 20 L V T M L S I F W F N V R E I 20 8074 23 M L S I F W F N V R E I S F N 20 8075 40 L S H M F F I K F F T V M E S 20 8076 43 M F F I K F F T V M E S S V L 20 8077 55 S V L L A M A F D R F V A V S 20 8078 57 L L A M A F D R F V A V S N P 20 8079 65 F V A V S N P L R Y A M I L T 20 8080 73 R Y A M I L T D S R I A Q I G 20 8081 80 D S R I A Q I G V A S V I R G 20 8082 92 I R G L L M L T P M V A L L I 20 8083 98 L T P M V A L L I R L S Y C H 20 8084 99 T P M V A L L I R L S Y C H S 20 8085 102 V A L L I R L S Y C H S Q V L 20 8086 105 L I R L S Y C H S Q V L H H S 20 8087 123 H P D V M K L S C T D T R I N 20 8088 126 V M K L S C T D T R I N S A V 20 8089 133 D T R I N S A V G L T A M F S 20 8090 146 F S T V G V D L L L I L L S Y 20 8091 148 T V G V D L L L I L L S Y V L 20 8092 150 G V D L L L I L L S Y V L I I 20 8093 152 D L L L I L L S Y V L I I R T 20 8094 155 L I L L S Y V L I I R T V L S 20 8095 164 I R T V L S V A S P E E R K E 20 8096 181 S T C V S H I V A F A I Y Y I 20 8097 184 V S H I V A F A I Y Y I P L I 20 8098 189 A F A I Y Y I P L I S L S I V 20 8099 192 I Y Y I P L I S L S I V H R F 20 8100 226 S P L M N P V I Y S V K T K Q 20 8101 238 T K Q I R R A V I K I L H S K 20 8102 11 T D L G L S I S T L V T M L S 18 8103 27 F W F N V R E I S F N A C L S 18 8104 47 K F F T V M E S S V L L A M A 18 8105 56 V L L A M A F D R F V A V S N 18 8106 61 A F D R F V A V S N P L R Y A 18 8107 77 I L T D S R I A Q I G V A S V 18 8108 127 M K L S C T D T R I N S A V G 18 8109 140 V G L T A M F S T V G V D L L 18 8110 168 L S V A S P E E R K E T F S T 18 8111 174 E E R K E T F S T C V S H I V 18 8112 178 E T F S T C V S H I V A E A I 18 8113 208 K Q A P A Y V H T M I A N T Y 18 8114 212 A Y V H T M I A N T Y L L I S 18 8115 213 Y V H T M I A N T Y L L I S P 18 8116 218 I A N T Y L L I S P L M N P V 18 8117 227 P L M N P V I Y S V K T K Q I 18 8118 235 S V K T K Q I R R A V I K I L 18 8119 1 M Y Y F L S M L S A T D L G L 16 8120 25 S I F W F N V R E I S F N A C 16 8121 33 E I S F N A C L S H M F F I K 16 8122 117 H H S Y C Y H P D V M K L S C 16 8123 187 I V A F A I Y Y I P L I S L S 16 8124 190 F A I Y Y I P L I S L S I V H 16 8125 203 V H R F G K Q A P A Y V H T M 16 8126 210 A P A Y V H T M I A N T Y L L 16 8127 5 L S M L S A T D L G L S I S T 14 8128 12 D L G L S I S T L V T M L S I 14 8129 17 I S T L V T M L S I F W F N V 14 8130 18 S T L V T M L S I F W F N V R 14 8131 21 V T M L S I F W F N V R E I S 14 8132 28 W F N V R E I S F N A C L S H 14 8133 48 F F T V M E S S V L L A M A F 14 8134 49 F T V M E S S V L L A M A F D 14 8135 53 E S S V L L A M A F D R F V A 14 8136 69 S N P L R Y A M I L T D S R I 14 8137 75 A M I L T D S R I A Q I G V A 14 8138 83 I A Q I G V A S V I R G L L M 14 8139 88 V A S V I R G L L M L T P M V 14 8140 89 A S V I R G L L M L T P M V A 14 8141 93 R G L L M L T P M V A L L I R 14 8142 94 G L L M L T P M V A L L I R L 14 8143 95 L L M L T P M V A L L I R L S 14 8144 103 A L L I R L S Y C H S Q V L H 14 8145 124 P D V M K L S C T D T R I N S 14 8146 137 N S A V G L T A M F S T V G V 14 8147 139 A V G L T A M F S T V G V D L 14 8148 142 L T A M F S T V G V D L L L I 14 8149 153 L L L I L L S Y V L I I R T V 14 8150 160 Y V L I I R T V L S V A S P E 14 8151 165 R T V L S V A S P E E R K E T 14 8152 185 S H I V A F A I Y Y I P L I S 14 8153 194 Y I P L I S L S I V H R F G K 14 8154 200 L S I V H R F G K Q A P A Y V 14 8155 214 V H T M I A N T Y L L I S P L 14 8156 215 H T M I A N T Y L L I S P L M 14 8157 221 T Y L L I S P L M N P V I Y S 14 8158 225 I S P L M N P V I Y S V K T K 14 8159 229 M N P V I Y S V K T K Q I R R 14 8160 6 S M L S A T D L G L S I S T L 12 8161 7 M L S A T D L G L S I S T L V 12 8162 8 L S A T D L G L S I S T L V T 12 8163 9 S A T D L Q L S I S T L V T M 12 8164 15 L S I S T L V T M L S I F W F 12 8165 29 F N V R E I S F N A C L S H M 12 8166 30 N V R E I S F N A C L S H M F 12 8167 32 R E I S F N A C L S H M F F I 12 8168 34 I S F N A C L S H M F F I K F 12 8169 39 C L S H M F F I K F F T V M E 12 8170 50 T V M E S S V L L A M A F D R 12 8171 66 V A V S N P L R Y A M I L T D 12 8172 72 L R Y A M I L T D S R I A Q I 12 8173 78 L T D S R I A Q I G V A S V I 12 8174 82 R I A Q I G V A S V I R G L L 12 8175 86 I G V A S V I R G L L M L T P 12 8176 90 S V I R G L L M L T P M V A L 12 8177 91 V I R Q L L M L T P M V A L L 12 8178 97 M L T P M V A L L I R L S Y C 12 8179 104 L L I R L S Y C H S Q V L H H 12 8180 109 S Y C H S Q V L H H S Y G Y H 12 8181 110 Y C H S Q V L H H S Y C Y H P 12 8182 115 V L H H S Y C Y H P D V M K L 12 8183 130 S C T D T R I N S A V G L T A 12 8184 134 T R I N S A V G L T A M F S T 12 8185 136 I N S A V G L T A M F S T V G 12 8186 138 S A V C L T A M F S T V G V D 12 8187 145 M F S T V G V D L L L I L L S 12 8188 147 S T V G V D L L L I L L S Y V 12 8189 156 I L L S Y V L I I R T V L S V 12 8190 173 P E E R K E T F S T C V S H I 12 8191 186 H I V A F A I Y Y I P L I S L 12 8192 196 P L I S L S I V H R F G K Q A 12 8193 201 S I V H R F G K Q A P A Y V H 12 8194 206 F G K Q A P A Y V H T M I A N 12 8195 207 G K Q A P A Y V H T M I A N T 12 8196 217 M I A N T Y L L I S P L M N P 12 8197 234 Y S V K T K Q I R R A V I K I 12 8198 240 Q I R R A V I K I L H S K E T 12 8199 26 I F W F N V R E I S F N A C L 11 8200 41 S H M F F I K F F T V M E S S 11 8201 107 R L S Y C H S Q V L H H S Y C 10 8202 101 M V A L L I R L S Y C H S Q V 9 8203 159 S Y V L I I R T V L S V A S P 9 8204 199 S L S I V H R F G K Q A P A Y 9 8205 233 I Y S V K T K Q I R R A V I K 9 8206 31 V R E I S F N A C L S H M F F 8 8207 37 N A C L S H M F F I K F F T V 8 8208 54 S S V L L A M A F D R F V A V 8 8209 63 D R F V A V S N P L R Y A M I 8 8210 112 H S Q V L H H S Y C Y H P D V 8 8211 113 S Q V L H H S Y C Y H P D V M 8 8212 154 L L I L L S Y V L I I R T V L 8 8213 167 V L S V A S P E E R K E T F S 8 8214 195 I P L I S L S I V H R F G K Q 8 8215 211 P A Y V H T M I A N T Y L L I 8 8216 220 N T Y L L I S P L M N P V I Y 8 8217 129 L S C T D T R I N S A V G L T 7 82i8 171 A S P E E R K E T F S T C V S 7 8219 3 Y F L S M L S A T D L G L S I 6 8220 13 L G L S I S T L V T M L S I F 6 8221 19 T L V T M L S I F W F N V R E 6 8222 22 T M L S I F W F N V R E I S F 6 8223 35 S F N A C L S H M F F I K F F 6 8224 36 F N A C L S H M F F I K F F T 6 8225 38 A C L S H M F F I K F F T V M 6 8226 44 F F I K F F T V M E S S V L L 6 8227 51 V M E S S V L L A M A F D R F 6 8228 52 M E S S V L L A M A F D R F V 6 8229 60 M A F D R F V A V S N P L R Y 6 8230 64 R F V A V S N P L R Y A M I L 6 8231 68 V S N P L R Y A M I L T D S R 6 8232 70 N P L R Y A M I L T D S R I A 6 8233 79 T D S R I A Q I G V A S V I R 6 8234 81 S R I A Q I C V A S V I R G L 6 8235 84 A Q I Q V A S V I R G L L M L 6 8236 96 L M L T P M V A L L I R L S Y 6 8237 100 P M V A L L I R L S Y C H S Q 6 8238 106 I R L S Y C H S Q V L H H S Y 6 8239 108 L S Y C H S Q V L H H S Y C Y 6 8240 111 C H S Q V L H H S Y C Y H P D 6 8241 116 L H H S Y C Y H P D V M K L S 6 8242 118 H S Y C Y H P D V M K L S G T 6 8243 120 Y C Y H P D V M K L S C T D T 6 8244 121 C Y H P D V M K L S C T D T R 6 8245 125 D V M K L S C T D T R I N S A 6 8246 128 K L S C T D T R I N S A V G L 6 8247 131 C T D T R I N S A V G L T A M 6 8248 132 T D T R I N S A V G L T A M F 6 8249 135 R I N S A V G L T A M F S T V 6 8250 144 A M F S T V C V D L L L I L L 6 8251 149 V G V D L L L T L L S Y V L I 6 8252 162 L I T R T V L S V A S P E E R 6 8253 166 T V L S V A S P E E R K E T F 6 8254 169 S V A S P E E R K E T F S T C 6 8255 172 S P E E R K E T F S T C V S H 6 8256 176 R K E T F S T C V S H I V A F 6 8257 180 F S T C V S H I V A F A I Y Y 6 8258 182 T C V S H I V A F A T Y Y I P 6 8259 183 C V S H I V A F A I Y Y I P L 6 8260 188 V A F A I Y Y I P L I S L S I 6 8261 193 Y Y I P L I S L S I V H R F G 6 8262 198 I S L S I V H R F G K Q A P A 6 8263 204 H R F G K Q A P A Y V H T M I 6 8264 205 R F G K Q A P A Y V H T M I A 6 8265 209 Q A P A Y V H T M I A N T Y L 6 8266 224 L I S P L M N P V I Y S V K T 6 8267 228 L M N P V I Y S V K T K Q I R 6 8268 232 V I Y S V K T K Q I R R A V I 6 8269 239 K Q I R R A V I K I L H S K E 6 8270 231 P V I Y S V K T K Q I R R A V 5 8271 58 L A M A F D R F V A V S N P L 1 8272 67 A V S N P L R Y A M I L T D S 1 8273 87 G V A S V I R G L L M L T P M 1 8274 122 Y H P D V M K L S C T D T R I 1 8275 170 V A S P E E R K E T F S T C V 1 8276 236 V K T K Q I R R A V I K I L H 1 8277 237 K T K Q I R R A V I K I L H S 1 8278 76 M I L T D S R I A Q I G V A S −5 8279 202 I V H R F G K Q A P A Y V H T −5 8280 HLA-DRB1*1101 15-mers v.1: 238P1B2 24 L S I F W F N V R E I S F N A 27 8281 157 L L S Y V L I I R T V L S V A 25 8282 178 E T F S T C V S H I V A F A I 23 8283 196 P L I S L S I V H R F G K Q A 23 8284 14 G L S I S T L V T M L S I F W 21 8285 85 Q I G V A S V I R G L L M L T 21 8286 139 A V G L T A M F S T V G V D L 21 8287 46 I K F F T V M E S S V L L A M 20 8288 98 L T P M V A L L I R L S Y C H 20 8289 99 T P M V A L L I R L S Y C H S 20 8290 197 L I S L S I V H R F G K Q A P 20 8291 200 L S I V H R F G K Q A P A Y V 20 8292 231 P V I Y S V K T K Q I R R A V 20 8293 92 I R G L L M L T P M V A L L I 19 8294 119 S Y C Y H P D V M K L S C T D 19 8295 148 T V G V D L L L I L L S Y V L 19 8296 158 L S Y V L I I R T V L S V A S 19 8297 89 A S V I R G L L M L T P M V A 18 8298 102 V A L L I R L S Y C H S Q V L 18 8299 161 V L I I R T V L S V A S P E E 18 8300 25 S I F W F N V R E I S F N A C 17 8301 45 F I K F F T V M E S S V L L A 17 8302 62 F D R F V A V S N P L R Y A M 17 8303 95 L L M L T P M V A L L I R L S 17 8304 143 T A M F S T V G V D L L L I L 17 8305 190 F A I Y Y I P L I S L S I V H 17 8306 229 M N P V I Y S V K T K Q T R R 17 8307 1 M Y Y F L S M L S A T D L G L 16 8308 71 P L R Y A M I L T D S R I A Q 16 8309 191 A I Y Y I P L I S L S I V H R 16 8310 219 A N T Y L L I S P L M N P V I 16 8311 7 M L S A T D L G L S I S T L V 15 8312 56 V L L A M A F D R F V A V S N 15 8313 83 I A Q I G V A S V I R G L L M 15 8314 110 Y C H S Q V L H H S Y C Y H P 15 8315 130 S C T D T R I N S A V G L T A 15 8316 160 Y V L I I R T V L S V A S P E 15 8317 162 L I I R T V L S V A S P E E R 15 8318 233 I Y S V K T K Q I R R A V I K 15 8319 235 S V K T K Q I R R A V I K I L 15 8320 239 K Q I R R A V I K I L H S K E 15 8321 18 S T L V T M L S I F W F N V R 14 8322 34 I S F N A C L S H M F F I K F 14 8323 54 S S V L L A M A F D R F V A V 14 8324 65 F V A V S N P L R Y A M I L T 14 8325 73 R Y A M I L T D S R I A Q I G 14 8326 74 Y A M I L T D S R I A Q I G V 14 8327 109 S Y C H S Q V L H H S Y C Y H 14 8328 120 Y C Y H P D V M K L S C T D T 14 8329 127 M K L S C T D T R I N S A V G 14 8330 133 D T R I N S A V G L T A M F S 14 8331 155 L I L L S Y V L I I R T V L S 14 8332 181 S T C V S H I V A F A I Y Y I 14 8333 5 L S M L S A T D L G L S I S T 13 8334 37 N A C L S H M F F I K F F T V 13 8335 40 L S H M F F I K F F T V M E S 13 8336 48 F F T V M E S S V L L A M A F 13 8337 151 V D L L L I L L S Y V L I I R 13 8338 152 D L L L I L L S Y V L I I R T 13 8339 177 K E T F S T C V S H I V A F A 13 8340 185 S H I V A F A I Y Y I P L I S 13 8341 192 I Y Y I P L I S L S I V H R F 13 8342 208 K Q A P A Y V H T M I A N T Y 13 8343 215 H T M I A N T Y L L I S P L M 13 8344 222 Y L L I S P L M N P V I Y S V 13 8345 226 S P L M N P V I Y S V K T K Q 13 8346 240 Q T R R A V T K I L H S K E T 13 8347 2 Y Y F L S M L S A T D L G L S 12 8348 17 I S T L V T M L S T F W F N V 12 8349 20 L V T M L S I F W F N V R E I 12 8350 28 W F N V R E I S F N A C L S H 12 8351 41 S H M F F I K F F T V M E S S 12 8352 43 M F F I K F F T V M E S S V L 12 8353 50 T V M E S S V L L A M A F D R 12 8354 59 A M A F D R F V A V S N P L R 12 8355 60 M A F D R F V A V S N P L R Y 12 8356 70 N P L R Y A M I L T D S R I A 12 8357 80 D S R I A Q I G V A S V I R G 12 8358 113 S Q V L H H S Y C Y H P D V M 12 8359 123 H P D V M K L S C T D T R I N 12 8360 136 I N S A V G L T A M F S T V G 12 8361 150 G V D L L L I L L S Y V L I I 12 8362 164 I R T V L S V A S P E E R K E 12 8363 189 A F A I Y Y I P L I S L S I V 12 8364 194 Y I P L I S L S I V H R F G K 12 8365 199 S L S I V H R F G K Q A P A Y 12 8366 211 P A Y V H T M I A N T Y L L I 12 8367 220 N T Y L L I S P L M N P V I Y 12 8368 230 N P V I Y S V K T K Q I R R A 12 8369 26 I F W F N V R E I S F N A C L 11 8370 187 I V A F A I Y Y I P L I S L S 11 8371 234 Y S V K T K Q I R R A V I K I 11 8372 33 E I S F N A C L S H M F F I K 10 8373 42 H M F F I K F F T V M E S S V 10 8374 107 R L S Y C H S Q V L H H S Y C 10 8375 117 H H S Y C Y H P D V M K L S C 10 8376 203 V H R F G K Q A P A Y V H T M 10 8377 207 G K Q A P A Y V H T M I A N T 10 8378 210 A P A Y V H T M I A N T Y L L 10 8379 44 F F I K F F T V M E S S V L L 9 8380 63 D R F V A V S N P L R Y A M I 9 8381 142 L T A M F S T V G V D L L L I 9 8382 154 L L I L L S Y V L I I R T V L 9 8383 238 T K Q I R R A V I K I L H S K 9 8384 15 L S I S T L V T M L S I F W F 8 8385 23 M L S I F W F N V R E I S F N 8 8386 39 C L S H M F F I K F F T V M E 8 8387 49 F T V M E S S V L L A M A F D 8 8388 58 L A M A F D R F V A V S N P L 8 8389 72 L R Y A M I L T D S R I A Q I 8 8390 75 A M I L T D S R I A Q I G V A 8 8391 86 I G V A S V I R G L L M L T P 8 8392 104 L L I R L S Y C H S Q V L H H 8 8393 108 L S Y C H S Q V L H H S Y C Y 8 8394 115 V L H H S Y C Y H P D V M K L 8 8395 126 V M K L S C T D T R I N S A V 8 8396 128 K L S C T D T R I N S A V G L 8 8397 146 F S T V G V D L L L I L L S Y 8 8398 156 I L L S Y V L I I R T V L S V 8 8399 167 V L S V A S P E E R K E T F S 8 8400 168 L S V A S P E E R K E T F S T 8 8401 169 S V A S P E E R K E T F S T C 8 8402 195 I P L I S L S I V H R F G K Q 8 8403 212 A Y V H T M I A N T Y L L T S 8 8404 217 M I A N T Y L L I S P L M N P 8 8405 225 I S P L M N P V I Y S V K T K 8 8406 227 P L M N P V I Y S V K T K Q I 8 8407 3 Y F L S M L S A T D L G L S I 7 8408 10 A T D L G L S I S T L V T M L 7 8409 11 T D L G L S I S T L V T M L S 7 8410 21 V T M L S I F W F N V R E I S 7 8411 30 N V R E I S F N A C L S H M F 7 8412 51 V M E S S V L L A M A F D R F 7 8413 55 S V L L A M A F D R F V A V S 7 8414 64 R F V A V S N P L R Y A M I L 7 8415 76 M I L T D S R I A Q I G V A S 7 8416 78 L T D S R I A Q I G V A S V I 7 8417 82 R I A Q I G V A S V I R G L L 7 8418 88 V A S V I R G L L M L T P M V 7 8419 94 G L L M L T P M V A L L I R L 7 8420 96 L M L T P M V A L L I R L S Y 7 8421 105 L I R L S Y C H S Q V L H H S 7 8422 121 C Y H P D V M K L S C T D T R 7 8423 145 M F S T V G V D L L L I L L S 7 8424 147 S T V G V D L L L I L L S Y V 7 8425 153 L L L I L L S Y V L I I R T V 7 8426 165 R T V L S V A S P E E R K E T 7 8427 174 E E R K E T F S T C V S H I V 7 8428 182 T C V S H I V A F A I Y Y I P 7 8429 186 H I V A F A I Y Y I P L I S L 7 8430 188 V A F A I Y Y I P L I S L S I 7 8431 214 V H T M I A N T Y L L I S P L 7 8432 218 I A N T Y L L I S P L M N P V 7 8433 223 L L I S P L M N P V I Y S V K 7 8434 4 F L S M L S A T D L G L S I S 6 8435 9 S A T D L G L S I S T L V T M 6 8436 12 D L G L S I S T L V T M L S I 6 8437 27 F W F N V R E I S F N A C L S 6 8438 31 V R E I S F N A C L S H M F F 6 8439 52 M E S S V L L A M A F D R F V 6 8440 53 E S S V L L A M A F D R F V A 6 8441 57 L L A M A F D R F V A V S N P 6 8442 66 V A V S N P L R Y A M I L T D 6 8443 69 S N P L R Y A M I L T D S R I 6 8444 77 I L T D S R I A Q I G V A S V 6 8445 79 T D S R I A Q I G V A S V I R 6 8446 90 S V I R G L L M L T P M V A L 6 8447 91 V I R G L L M L T P M V A L L 6 8448 93 R G L L M L T P M V A L L I R 6 8449 100 P M V A L L I R L S Y C H S Q 6 8450 101 M V A L L I R L S Y C H S Q V 6 8451 103 A L L I R L S Y C H S Q V L H 6 8452 112 H S Q V L H H S Y C Y H P D V 6 8453 124 P D V M K L S C T D T R I N S 6 8454 134 T R I N S A V G L T A M F S T 6 8455 137 N S A V G L T A M F S T V G V 6 8456 144 A M F S T V G V D L L L I L L 6 8457 149 V G V D L L L I L L S Y V L I 6 8458 159 S Y V L I I R T V L S V A S P 6 8459 170 V A S P E E R K E T F S T C V 6 8460 180 F S T C V S H I V A F A I Y Y 6 8461 184 V S H I V A F A I Y Y I P L I 6 8462 201 S I V H R F G K Q A P A Y V H 6 8463 216 T M I A N T Y L L I S P L M N 6 8464 221 T Y L L I S P L M N P V I Y S 6 8465 224 L I S P L M N P V I Y S V K T 6 8466 61 A F D R F V A V S N P L R Y A 4 8467 81 S R I A Q I G V A S V I R G L 3 8468 84 A Q I G V A S V I R G L L M L 3 8469 97 M L T P M V A L L I R L S Y C 3 8470 16 S I S T L V T M L S I F W F N 2 8471 22 T M L S I F W F N V R E I S F 2 8472 125 D V M K L S C T D T R I N S A 2 8473 163 I I R T V L S V A S P E E R K 2 8474 166 T V L S V A S P E E R K E T F 2 8475 175 E R K E T F S T C V S H I V A 2 8476 198 I S L S I V H R F G K Q A P A 2 8477 232 V I Y S V K T K Q I R R A V I 2 8478 32 R E I S F N A C L S H M F F I 1 8479 36 F N A C L S H M F F I K F F T 1 8480 47 K F F T V M E S S V L L A M A 1 8481 67 A V S N P L R Y A M I L T D S 1 8482 68 V S N P L R Y A M I L T D S R 1 8483 106 I R L S Y C H S Q V L H H S Y 1 8484 116 L H H S Y C Y H P D V M K L S 1 8485 118 H S Y C Y H P D V M K L S C T 1 8486 132 T D T R I N S A V G L T A M F 1 8487 135 R I N S A V G L T A M F S T V 1 8488 141 G L T A M F S T V G V D L L L 1 8489 176 R K E T F S T C V S H I V A F 1 8490 179 T F S T C V S H I V A F A I Y 1 8491 183 C V S H I V A F A I Y Y I P L 1 8492 193 Y Y I P L I S L S I V H R F G 1 8493 202 I V H R F G P Q A P A Y V H T 8494

TABLE XIXC part 2 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID H2-Ak 15-mers, v.1B 2 I T S T L Q N I T S T S I I F 16 8495 15 I F L L T G V P G L E A F H T 16 8496 48 L I L F A T I T Q P S L H E P 14 8497 53 T I T Q P S L H E P M Y Y F L 14 8498 61 E P M Y Y F L S M L S A T D L 14 8499 43 L L G N S L I L F A T I T Q P 12 8500 12 T S I I F L L T G V P G L E A 10 8501 21 V P G L E A F H T W I S I P F 10 8502 47 S L I L F A T I T Q P S L H E 10 8503 8 N I T S T S I I F L L T G V P 8 8504 9 I T S T S I I F L L T G V P G 8 8505 11 S T S I I F L L T G V P G L E 8 8506 17 L L T G V P G L E A F H T W I 8 8507 18 L T G V P G L E A F H T W I S 8 8508 28 H T W I S I P F C F L S V T A 8 8509 31 I S I P F C F L S V T A L L G 8 8510 34 P F C F L S V T A L L G N S L 8 8511 36 C F L S V T A L L G N S L I L 8 8512 37 F L S V T A L L G N S L I L F 8 8513 41 T A L L G N S L I L F A T I T 8 8514 42 A L L G N S L I L F A T I T Q 8 8515 44 L G N S L I L F A T I T Q P S 8 8516 57 P S L H E P M Y Y F L S M L S 8 8517 60 H E P M Y Y F L S M L S A T D 8 8518 3 T S T L Q N I T S T S I I F L 6 8519 5 T L Q N I T S T S I I F L L T 6 8520 25 E A F H T W I S I P F C F L S 6 8521 27 F H T W I S I P F C F L S V T 6 8522 32 S I P F C F L S V T A L L G N 6 8523 38 L S V T A L L G N S L I L F A 6 8524 40 V T A L L G N S L I L F A T I 6 8525 45 G N S L I L F A T I T Q P S L 4 8526 1 F I T S T L Q N I T S T S I I 2 8527 4 S T L Q N I T S T S I I F L L 2 8528 30 W I S I P F C F L S V T A L L 2 8529 33 I P F C F L S V T A L L G N S 2 8530 62 P M Y Y F L S M L S A T D L G 2 8531 H2-Ek 15-mers, v.1B 12 T S I I F L L T G V P G L E A 20 8532 15 I F L L T G V P G L E A F H T 20 8533 18 L T G V P G L E A F H T W I S 20 8534 37 F L S V T A L L G N S L I L F 20 8535 3 T S T L Q N I T S T S I I F L 16 8536 11 S T S I I F L L T G V P G L E 16 8537 21 V P G L E A F H T W I S I P F 16 8538 34 P F C F L S V T A L L G N S L 16 8539 41 T A L L G N S L I L F A T I T 16 8540 45 G N S L I L F A T I T Q P S L 16 8541 48 L I L F A T I T Q P S L H E P 16 8542 27 F H T W I S I P F C F L S V T 12 8543 32 S I P F C F L S V T A L L G N 12 8544 61 E P M Y Y F L S M L S A T D L 12 8545 62 P M Y Y F L S M L S A T D L G 12 8546 6 L Q N I T S T S I I F L L T G 10 8547 14 I I F L L T G V P G L E A F H 10 8548 28 H T W I S I P F C F L S V T A 10 8549 30 W I S I P F C F L S V T A L L 10 8550 35 F C F L S V T A L L G N S L I 10 8551 38 L S V T A L L G N S L I L F A 10 8552 40 V T A L L G N S L I L F A T I 10 8553 44 L G N S L I L F A T I T Q P S 10 8554 46 N S L I L F A T I T Q P S L H 10 8555 47 S L I L F A T I T Q P S L H E 10 8556 51 F A T I T Q P S L H E P M Y Y 10 8557 53 T I T Q P S L H E P M Y Y F L 10 8558 56 Q P S L H E P M Y Y F L S M L 10 8559 5 T L Q N I T S T S I I F L L T 6 8560 7 Q N I T S T S I I F L L T G V 6 8561 8 N I T S T S I I F L L T G V P 6 8562 9 I T S T S I I F L L T G V P G 6 8563 10 T S T S I I F L L T G V P G L 6 8564 13 S I I F L L T G V P G L E A F 6 8565 24 L E A F H T W I S I P F C F L 6 8566 25 E A F H T W I S I P F C F L S 6 8567 26 A F H T W I S I P F C F L S V 6 8568 29 T W I S I P F C F L S V T A L 6 8569 31 I S I P F C F L S V T A L L G 6 8570 33 I P F C F L S V T A L L G N S 6 8571 42 A L L G N S L I L F A T I T Q 6 8572 43 L L G N S L I L F A T I T Q P 6 8573 52 A T I T Q P S L H E P M Y Y F 6 8574 60 H E P M Y Y F L S M L S A T D 6 8575 17 L L T G V P G L E A F H T W I 4 8576 HLA-DRB1*0101 15-mers, v.1B: 238P1B2 37 F L S V T A L L G N S L I L F 33 8577 11 S T S I I F L L T G V P G L E 31 8578 15 I F L L T Q V P G L E A F H T 31 8579 3 T S T L Q N I T S T S I I F L 29 8580 32 S I P F C F L S V T A L L G N 28 8581 61 E P M Y Y F L S M L S A T D L 27 8582 12 T S I I F L L T G V P G L E A 25 8583 18 L T G V P G L E A F H T W I S 25 8584 24 L E A F H T W I S I P F C F L 24 8585 27 F H T W I S I P F C F L S V T 24 8586 34 P F C F L S V T A L L G N S L 24 8587 48 L I L F A T I T Q P S L H E P 24 8588 13 S I I F L L T G V P G L E A F 22 8589 21 V P G L E A F H T W I S I P F 22 8590 47 S L I L F A T I T Q P S L H E 22 8591 60 H E P M Y Y F L S M L S A T D 22 8592 44 L G N S L I L F A T I T Q P S 20 8593 28 H T W I S I P F C F L S V T A 19 8594 4 S T L Q N I T S T S I I F L L 18 8595 38 L S V T A L L G N S L I L F A 18 8596 46 N S L I L F A T I T Q P S L H 18 8597 6 L Q N I T S T S I I F L L T G 17 8598 26 A F H T W I S I P F C F L S V 17 8599 51 F A T I T Q P S L H E P M Y Y 17 8600 40 V T A L L G N S L I L F A T I 16 8601 41 T A L L G N S L I L F A T I T 16 8602 56 Q P S L H E P M Y Y F L S M L 16 8603 33 I P F C F L S V T A L L G N S 15 8604 39 S V T A L L G N S L I L F A T 15 8605 43 L L G N S L I L F A T I T Q P 15 8606 45 G N S L I L F A T I T Q P S L 15 8607 2 I T S T L Q N I T S T S I I F 14 8608 5 T L Q N I T S T S I I F L L T 14 8609 10 T S T S I I F L L T G V P G L 14 8610 14 I I F L L T G V P G L E A F H 14 8611 31 I S I P F C F L S V T A L L G 14 8612 57 P S L H E P M Y Y F L S M L S 14 8613 50 L F A T I T Q P S L H E P M Y 12 8614 62 P M Y Y F L S M L S A T D L G 11 8615 35 F C F L S V T A L L G N S L I 10 8616 49 I L F A T I T Q P S L H E P M 10 8617 1 F I T S T L Q N I T S T S I I 9 8618 9 I T S T S I I F L L T G V P G 9 8619 16 F L L T G V P G L E A F H T W 9 8620 20 G V P G L E A F H T W I S I P 9 8621 22 P G L E A F H T W I S I P F C 9 8622 30 W I S I P F C F L S V T A L L 9 8623 36 C F L S V T A L L G N S L I L 9 8624 42 A L L G N S L I L F A T I T Q 9 8625 58 S L H E P M Y Y F L S M L S A 9 8626 7 Q N I T S T S I I F L L T G V 8 8627 23 G L E A F H T W I S I P F C F 8 8628 29 T W I S I P F C F L S V T A L 8 8629 52 A T I T Q P S L H E P M Y Y F 8 8630 53 T I T Q P S L H E P M Y Y F L 8 8631 54 I T Q P S L H E P M Y Y F L S 8 8632 55 T Q P S L H E P M Y Y F L S M 8 8633 59 L H E P M Y Y F L S M L S A T 8 8634 8 N I T S T S I I F L L T G V P 7 8635 25 E A F H T W I S I P F C F L S 7 8636 17 L L T G V P G L E A F H T W I 1 8637 19 T G V P G L E A F H T W I S I 1 8638 HLA-DRB1*0301 (DR17) 15-mers, v.1B: 238P1B2 40 V T A L L G N S L I L F A T I 22 8639 48 L I L F A T I T Q P S L H E P 22 8640 6 L Q N I T S T S I I F L L T G 21 8641 37 F L S V T A L L G N S L I L F 21 8642 13 S I I F L L T G V P G L E A F 20 8643 56 Q P S L H E P M Y Y F L S M L 19 8644 32 S I P F C F L S V T A L L C N 18 8645 24 L E A F H T W I S I P F C F L 16 8646 53 T I T Q P S L H E P M Y Y F L 15 8647 14 I I F L L T G V P G L E A F H 13 8648 15 I F L L T G V P G L E A F H T 13 8649 34 P F C F L S V T A L L G N S L 13 8650 11 S T S I I F L L T G V P G L E 12 8651 12 T S I I F L L T G V P G L E A 12 8652 39 S V T A L L G N S L I L F A T 12 8653 41 T A L L G N S L I L F A T I T 12 8654 45 G N S L I L F A T I T Q P S L 12 8655 46 N S L I L F A T I T Q P S L H 12 8656 51 F A T I T Q P S L H E P M Y Y 12 8657 60 H E P M Y Y F L S M L S A T D 12 8658 18 L T G V P G L E A F H T W I S 11 8659 27 F H T W I S I P F C F L S V T 11 8660 28 H T W I S I P F C F L S V T A 11 8661 33 I P F C F L S V T A L L G N S 11 8662 35 F C F L S V T A L L G N S L I 11 8663 47 S L I L F A T I T Q P S L H E 11 8664 55 T Q P S L H E P M Y Y F L S M 11 8665 3 T S T L Q N I T S T S I I F L 10 8666 16 F L L T G V P G L E A F H T W 10 8667 17 L L T G V P G L E A F H T W I 10 8668 21 V P G L E A F H T W I S I P F 10 8669 30 W I S I P F C F L S V T A L L 10 8670 54 I T Q P S L H E P M Y Y F L S 10 8671 5 T L Q N I T S T S I I F L L T 9 8672 7 Q N I T S T S I I F L L T G V 9 8673 38 L S V T A L L G N S L I L F A 9 8674 26 A F H T W I S I P F C F L S V 8 8675 59 L H E P M Y Y F L S M L S A T 8 8676 8 N I T S T S I I F L L T G V P 4 8677 20 G V P G L E A F H T W I S I P 3 8678 29 T W I S I P F C F L S V T A L 3 8679 36 C F L S V T A L L G N S L I L 3 8680 43 L L G N S L I L F A T I T Q P 3 8681 50 L F A T I T Q P S L H E P M Y 3 8682 1 F I T S T L Q N I T S T S I I 2 8683 2 I T S T L Q N I T S T S I I F 2 8684 10 T S T S I I F L L T G V P G L 2 8685 25 E A F H T W I S I P F C F L S 2 8686 31 I S I P F C F L S V T A L L G 2 8687 42 A L L G N S L I L F A T I T Q 2 8688 44 L G N S L I L F A T I T Q P S 2 8689 49 I L F A T I T Q P S L H E P M 2 8690 52 A T I T Q P S L H E P M Y Y F 2 8691 57 P S L H E P M Y Y F L S M L S 2 8692 58 S L H E P M Y Y F L S M L S A 2 8693 61 E P M Y Y F L S M L S A T D L 2 8694 62 P M Y Y F L S M L S A T D L G 2 8695 4 S T L Q N I T S T S I I F L L 1 8696 9 I T S T S I I F L L T G V P G 1 8697 19 T G V P G L E A F H T W I S I 1 8698 HLA-DRB1*0401 (DR4Dw4) 15-mers, v.1B: 238P1B2 48 L I L F A T I T Q P S L H E P 28 8699 3 T S T L Q N I T S T S I I F L 26 8700 21 V P G L E A F H T W I S I P F 26 8701 45 G N S L I L F A T I T Q P S L 26 8702 60 H E P M Y Y F L S M L S A T D 26 8703 27 F H T W I S I P F C F L S V T 22 8704 32 S I P F C F L S V T A L L G N 22 8705 61 E P M Y Y F L S M L S A T D L 22 8706 11 S T S I I F L L T G V P G L E 20 8707 15 I F L L T G V P G L E A F H T 20 8708 18 L T G V P C L E A F H T W I S 20 8709 37 F L S V T A L L G N S L I L F 20 8710 47 S L I L F A T I T Q P S L H E 20 8711 20 C V P G L E A F H T W I S I P 18 8712 31 I S I P F C F L S V T A L L G 18 8713 38 L S V T A L L G N S L I L F A 18 8714 13 S I I F L L T G V P G L E A F 16 8715 24 L E A F H T W I S I P F C F L 16 8716 34 P F C F L S V T A L L G N S L 16 8717 62 P M Y Y F L S M L S A T D L G 16 8718 6 L Q N I T S T S I I F L L T G 14 8719 12 T S I I F L L T G V P G L E A 14 8720 28 H T W I S I P F C F L S V T A 14 8721 30 W I S I P F C F L S V T A L L 14 8722 35 F C F L S V T A L L G N S L I 14 8723 41 T A L L G N S L I L F A T I T 14 8724 46 N S L I L F A T I T Q P S L H 14 8725 56 Q P S L H E P M Y Y F L S M L 14 8726 2 I T S T L Q N I T S T S I I F 12 8727 4 S T L Q N I T S T S I I F L L 12 8728 8 N I T S T S I I F L L T G V P 12 8729 10 T S T S I I F L L T G V P G L 12 8730 29 T W I S I P F C F L S V T A L 12 8731 33 I P F C F L S V T A L L G N S 12 8732 36 C F L S V T A L L G N S L I L 12 8733 39 S V T A L L C N S L I L F A T 12 8734 42 A L L G N S L I L F A T I T Q 12 8735 43 L L G N S L I L F A T I T Q P 12 8736 44 L G N S L I L F A T I T Q P S 12 8737 52 A T I T Q P S L H E P M Y Y F 12 8738 14 I I F L L T G V P G L E A F H 8 8739 40 V T A L L G N S L I L F A T I 8 8740 51 F A T I T Q P S L H E P M Y Y 8 8741 1 F I T S T L Q N I T S T S I I 6 8742 5 T L Q N I T S T S I I F L L T 6 8743 7 Q N I T S T S I I F L L T G V 6 8744 9 I T S T S I I F L L T G V P G 6 8745 19 T G V P G L E A F H T W I S I 6 8746 22 P G L E A F H T W I S I P F C 6 8747 25 E A F H T W I S I P F C F L S 6 8748 50 L F A T I T Q P S L H E P M Y 6 8749 53 T I T Q P S L H E P M Y Y F L 6 8750 55 T Q P S L H E P M Y Y F L S M 6 8751 57 P S L H E P M Y Y F L S M L S 6 8752 58 S L H E P M Y Y F L S M L S A 6 8753 59 L H E P M Y Y F L S M L S A T 6 8754 HLA-DRB1*1101 15-mers, v.1B: 238P1B2 34 P F C F L S V T A L L G N S L 23 8755 61 E P M Y Y F L S M L S A T D L 22 8756 15 I F L L T G V P G L E A F H T 20 8757 11 S T S I I F L L T G V P G L E 19 8758 12 T S I I F L L T G V P G L E A 19 8759 27 F H T W I S I P F C F L S V T 16 8760 32 S I P F C F L S V T A L L G N 16 8761 48 L I L F A T I T Q P S L H E P 16 8762 37 F L S V T A L L G N S L I L F 14 8763 51 F A T I T Q P S L H E P M Y Y 14 8764 21 V P G L E A F H T W I S I P F 13 8765 28 H T W I S I P F C F L S V T A 13 8766 47 S L I L F A T I T Q P S L H E 13 8767 57 P S L H E P M Y Y F L S M L S 13 8768 60 H E P M Y Y F L S M L S A T D 13 8769 3 T S T L Q N I T S T S I I F L 12 8770 9 I T S T S I T F L L T G V P G 12 8771 18 L T G V P C L E A F H T W I S 12 8772 41 T A L L G N S L I L F A T I T 12 8773 46 N S L I L F A T I T Q P S L H 12 8774 62 P M Y Y F L S M L S A T D L G 11 8775 13 S I T F L L T G V P G L E A F 10 8776 24 L E A F H T W I S T P F C F L 10 8777 8 N I T S T S I I F L L T G V P 9 8778 14 I I F L L T G V P G L E A F H 9 8779 6 L Q N I T S T S I I F L L T G 8 8780 20 G V P G L E A F H T W I S I P 8 8781 23 C L E A F H T W I S I P F C F 8 8782 25 E A F H T W I S I P F C F L S 8 8783 52 A T I T Q P S L H E P M Y Y F 8 8784 30 W I S I P F C F L S V T A L L 7 8785 31 I S I P F C F L S V T A L L G 7 8786 35 F G F L S V T A L L G N S L I 7 8787 38 L S V T A L L G N S L I L F A 7 8788 40 V T A L L G N S L I L F A T I 7 8789 44 L G N S L I L F A T I T Q P S 7 8790 2 I T S T L Q N I T S T S I I F 6 8791 36 C F L S V T A L L G N S L I L 6 8792 42 A L L G N S L I L F A T I T Q 6 8793 43 L L G N S L I L F A T I T Q P 6 8794 45 G N S L I L F A T I T Q P S L 6 8795 53 T I T Q P S L H E P M Y Y F L 6 8796 56 Q P S L H E P M Y Y F L S M L 6 8797 33 I P F C F L S V T A L L G N S 4 8798 49 I L F A T I T Q P S L H E P M 3 8799 1 F I T S T L Q N I T S T S I I 2 8800 4 S T L Q N I T S T S I I F L L 1 8801 5 T L Q N I T S T S I I F L L T 1 8802 7 Q N I T S T S I I F L L T G V 1 8803 16 F L L T G V P G L E A F H T W 8804

TABLE XIXC part 3 Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ ID HLA-DRB1*0101 15-mers, v.2: 238P1B2 7 M I A N T Y L L T S P L M N P 23 8805 8 I A N T Y L L T S P L M N P V 23 8806 1 P A Y V H T M I A N T Y L L T 22 8807 2 A Y V H T M T A N T Y L L T S 17 8808 3 Y V H T M I A N T Y L L T S P 17 8809 9 A N T Y L L T S P L M N P V I 17 8810 11 T Y L L T S P L M N P V I Y S 17 8811 5 H T M I A N T Y L L T S P L M 16 8812 10 N T Y L L T S P L M N P V I Y 16 8813 12 Y L L T S P L M N P V I Y S V 16 8814 15 T S P L M N P V I Y S V K T K 16 8815 13 L L T S P L M N P V I Y S V K 13 8816 6 T M I A N T Y L L T S P L M N 10 8817 14 L T S P L M N P V I Y S V K T 9 8818 4 V H T M I A N T Y L L T S P L 8 8819 HLA-DRB1*0301 (DR17) 15-mers, v.2: 238P1B2 1 P A Y V H T M I A N T Y L L T 19 8820 2 A Y V H T M I A N T Y L L T S 16 8821 10 N T Y L L T S P L M N P V I Y 14 8822 11 T Y L L T S P L M N P V I Y S 14 8823 4 V H T M I A N T Y L L T S P L 13 8824 5 H T M I A N T Y L L T S P L M 11 8825 7 M I A N T Y L L T S P L M N P 10 8826 13 L L T S P L M N P V I Y S V K 10 8827 15 T S P L M N P V I Y S V K T K 10 8828 3 Y V H T M I A N T Y L L T S P 9 8829 12 Y L L T S P L M N P V I Y S V 7 8830 9 A N T Y L L T S P L M N P V I 5 8831 14 L T S P L M N P V I Y S V K T 4 8832 8 I A N T Y L L T S P L M N P V 2 8833 6 T M I A N T Y L L T S P L M N 1 8834 HLA-DRB1*0401 (DR4Dw4) 15-mers, v.2: 238P1B2 2 A Y V H T M I A N T Y L L T S 18 8835 3 Y V H T M I A N T Y L L T S P 18 8836 7 M I A N T Y L L T S P L M N P 18 8837 8 I A N T Y L L T S P L M N P V 18 8838 12 Y L L T S P L M N P V I Y S V 18 8839 9 A N T Y L L T S P L M N P V I 16 8840 4 V H T M I A N T Y L L T S P L 14 8841 5 H T M I A N T Y L L T S P L M 14 8842 11 T Y L L T S P L M N P V I Y S 14 8843 15 T S P L M N P V I Y S V K T K 14 8844 1 P A Y V H T M I A N T Y L L T 8 8845 10 N T Y L L T S P L M N P V I Y 8 8846 14 L T S P L M N P V I Y S V K T 6 8847 HLA-DRB1*1101 15-mers, v.2: 238P1B2 1 P A Y V H T M I A N T Y L L T 12 8848 5 H T M I A N T Y L L T S P L M 12 8849 10 N T Y L L T S P L M N P V I Y 12 8850 9 A N T Y L L T S P L M N P V I 10 8851 2 A Y V H T M I A N T Y L L T S 8 8852 7 M I A N T Y L L T S P L M N P 8 8853 11 T Y L L T S P L M N P V I Y S 8 8854 15 T S P L M N P V I Y S V K T K 8 8855 4 V H T M I A N T Y L L T S P L 7 8856 8 I A N T Y L L T S P L M N P V 7 8857 12 Y L L T S P L M N P V I Y S V 7 8858 13 L L T S P L M N P V I Y S V K 7 8859 6 T M I A N T Y L L T S P L M N 6 8860 14 L T S P L M N P V I Y S V K T 8861

TABLE XX Frequently Occurring Motifs avrg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleic acid-binding protein functions as transcription factor, nuclear location probable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase, generate superoxide terminal)/b6/petB ig 19% Immunoglobulin domain domains are one hundred amino acids long and include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandem repeats of about 40 residues, each containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub-membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions pkinase 23% Protein kinase domain conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins rvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton oxidored_q1 32% NADH- membrane associated. Involved in proton Ubiquinone/plastoquinone translocation across the membrane (complex I), various chains efhand 24% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha- helical domain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases, centered on a catalytic aspartyl residue Collagen 42% Collagen triple helix repeat extracellular structural proteins involved in (20 copies) formation of connective tissue. The sequence consists of the G-X-Y and the polypeptide chains forms a triple helix. fn3 20% Fibronectin type III domain Located in the extracellular ligand-binding region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane receptor seven hydrophobic transmembrane regions, with (rhodopsin family) the N-terminus located extracellularly while the C-terminus is cytoplasmic. Signal through G proteins

TABLE XXI Motifs and Post-translational Modifications of 238P1B2 cAMP- and cGMP-dependent protein kinase phosphorylation site. 176-179 RKeT (SEQ ID: 8862) Protein kinase C phosphorylation site. 235-237 SvK Casein kinase II phosphorylation site.  9-12 SatD (SEQ ID: 8863) 50-53 TvmE (SEQ ID: 8864) 130-133 SctD (SEQ ID: 8865) 172-175 SpeE (SEQ ID: 8866) N-myristoylation site. 14-19 GLsiST (SEQ ID: 8867) G-protein coupled receptors family 1 signature. 52-68 MESsvLlaMAFDRFvaV (SEQ ID: 8868) G-protein coupled receptors family 1  1-234

TABLE XXII Physical Features of 238P1B2 Bioinformatic Program Outcome 238P1B2 Variant 1A ORF ORF finder 3758 bp Protein length 254 aa Transmembrane TM Pred N terminus intracellular, 6 TM region TM helices at 3-29, 44-62, 86-110, 144-161, 182-203, 217-236aa HMMTop N terminus intracellular, 6TM TM helices at 6-28, 43-62, 86-105, 136-158, 180-203, 216-235aa Sosui Membrane protein, 6TM TM helices at 6-28, 42-64, 87-109, 144-166, 187-209, 217-237aa TMHMM N terminus intracellular, 6TM TM helices at 7-29, 44-66, 86-108, 142-164, 185-207, 212-234aa Signal Peptide Signal P cleavage site between 169-170aa pI pI/MW tool pI 9.24 Molecular weight pI/MW tool 28.59 kDa Localization PSORT 60% plasma membrane, 42.9% mitochondrial inner membrane, 40% Golgi, 30% endoplasmic reticulum membrane PSORT II 44.4% endoplasmic reticulum, 22.2% plasma membrane Motifs Pfam 7TM receptor (rhodopsin family) Prints Olfactory receptor signature, Type III secretion system inner membrane R protein, fibronectin Type III repeat signature Blocks no significant motif 238P1B2 Variant 1B ORF ORF finder 3758 bp Protein length 316 aa Transmembrane TM Pred N terminus extracellular, 8 TM region TM helices at 9-27, 33-51, 75-91, 98-125, 145-171, 206-226, 249-265, 276-296aa HMMTop N terminus extracellular, 7TM TM helices at 29-53, 66-90, 105-124, 148-171, 202-226, 241-265, 278-297aa Sosui Membrane protein, 8TM TM helices at 2-24, 33-55, 67-89, 104-126, 149-171, 206-228, 245-267, 279-299aa TMHMM N terminus intracellular, 7TM TM helices at 30-52, 65-87, 102-124, 145-167, 204-226, 247-269, 274-296aa Signal Peptide Signal P cleavage site between aa 26 and 27 pI pI/MW tool pI 9.03 Molecular weight pI/MW tool 35.34 kDa Localization PSORT 64% plasma membrane, 46% Golgi, 37% endoplasmic reticulum membrane PSORT II 44.4% endoplasmic reticulum, 33.3% plasma membrane Motifs Pfam 7TM receptor (rhodopsin family), polysaccharide biosynthesis protein Prints Olfactory receptor signature, Type III secretion system inner membrane R protein, fibronectin Type III repeat signature Blocks no significant motif

TABLE XXIII Exon compositions of 238P1B2 v.1 Exon Number Start End Exon 1 1 3758 

1. A composition comprising an isolated 238P1B2 protein comprising the amino acid sequence of SEQ ID NO:8873.
 2. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
 3. A pharmaceutical composition that comprises the composition of claim 1 in a human unit dose form. 